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Beta-glucan: An ideal immunostimulant in aquaculture (a review)

  • ICAR-Central Inland Fisheries Research Institute
  • Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur
  • College of Fisheries, Central Agricultural University

Abstract and Figures

The major hindrance in the development and sustainability of aquaculture industry is the occurrence of various diseases in the farming systems. Today, preventive and management measures are central concern to overcome such outbreak of diseases. Immunostimulants are considered as an effective tool for enhancing immune status of cultured organisms. Among different immunostimulants used in aquaculture practices, β-glucan is one of the promising immunostimulant, which is a homopolysaccharide of glucose molecule linked by the glycoside bond. It forms the major constituents of cell wall of some plants, fungi, bacteria, mushroom, yeast, and seaweeds. Major attention on β-glucan was captivated with the gain in knowledge on its receptors and the mechanism of action. The receptor present inside the animal body recognizes and binds to β-glucan, which in turn renders the animal with high resistance and enhanced immune response. This review highlights β-glucan as an immunostimulant, its effective dosages, and route of administration and furthermore provides an outline on role of β-glucan in enhancing growth, survival, and protection against infectious pathogens pertaining to fishes and shellfishes. Study also summarizes the effect of β-glucan on its receptors, recognition of proteins, immune-related enzymes, immune-related gene expression and their mechanisms of action.
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Fish Physiology and Biochemistry
ISSN 0920-1742
Volume 39
Number 3
Fish Physiol Biochem (2013) 39:431-457
DOI 10.1007/s10695-012-9710-5
Beta-glucan: an ideal immunostimulant in
aquaculture (a review)
D.K.Meena, Pronob Das, Shailesh
Kumar, S.C.Mandal, A.K.Prusty,
S.K.Singh, M.S.Akhtar, B.K.Behera,
Kundan Kumar, et al.
1 23
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Beta-glucan: an ideal immunostimulant in aquaculture
(a review)
D. K. Meena
Pronob Das
Shailesh Kumar
S. C. Mandal
A. K. Prusty
S. K. Singh
M. S. Akhtar
B. K. Behera
Kundan Kumar
A. K. Pal
S. C. Mukherjee
Received: 6 February 2012 / Accepted: 28 August 2012 / Published online: 11 September 2012
Ó Springer Science+Business Media B.V. 2012
Abstract The major hindrance in the development
and sustainability of aquaculture industry is the
occurrence of various diseases in the farming systems.
Today, preventive and management measures are
central concern to overcome such outbreak of dis-
eases. Immunostimulants are considered as an effec-
tive tool for enhancing immune status of cultured
organisms. Among different immunostimulants used
in aquaculture practices, b-glucan is one of the
promising immunostimulant, which is a homopoly-
saccharide of glucose molecule linked by the glyco-
side bond. It forms the major constituents of cell wall
of some plants, fungi, bacteria, mushroom, yeast, and
seaweeds. Major attention on b-glucan was captivated
with the gain in knowledge on its receptors and the
mechanism of action. The receptor present inside the
animal body recognizes and binds to b-glucan, which
in turn renders the animal with high resistance and
enhanced immune response. This review highlights
b-glucan as an immunostimulant, its effective dos-
ages, and route of administration and furthermore
provides an outline on role of b-glucan in enhancing
growth, survival, and protection against infectious
pathogens pertaining to fishes and shellfishes. Study
also summarizes the effect of b-glucan on its recep-
tors, recognition of proteins, immune-related
enzymes, immune-related gene expression and their
mechanisms of action.
Keywords b-Glucan b-Glucan receptor b-Glucan
binding protein Prophenoloxidase
Immunostimulant Aquaculture Prebiotics
D. K. Meena, Pronob Das, Shailesh Kumar have contributed
equally to this work.
D. K. Meena P. Das (&) B. K. Behera
Central Inland Fisheries Research Institute, Barracklpore,
Kolkata 700120, West Bengal, India
S. Kumar S. K. Singh K. Kumar
A. K. Pal S. C. Mukherjee
Central Institute of Fisheries Education, Seven Bungalow,
Versova, Mumbai 400061, India
S. C. Mandal
College of Fisheries, Central Agricultural University,
Lembucherra 799210, Tripura, India
A. K. Prusty
Project Directorate for Farming System Research
(PDFSR), Modipuram, Meerut 250110, UP, India
M. S. Akhtar
Directorate of Coldwater Fisheries Research, Bhimtal,
Nainital 263136, Uttarakhand, India
Fish Physiol Biochem (2013) 39:431–457
DOI 10.1007/s10695-012-9710-5
Author's personal copy
BG b-Glucan
LGBP Lipopolysaccharide and b-glucan
binding protein
proPO Prophenoloxidase
BGR b-Glucan receptor
LPS Lipopolysaccharide
TLR Toll like receptor
WSSV White spot syndrome virus
RPS Relative percent survival
FED Feed efficiency ratio
YYS Yeast and yeast subcomponents
YCW Yeast cell wall
YBG Yeast b-glucan
BYG Brewer’s yeast glucan
CMBG Carboxymethyl b-glucan
THC Total hemocyte count
BGBP-HDL b-Glucan binding protein–high density
Intensification of aquaculture practices leads to the
emergence of several pathogenic organisms. Rapid and
uncontrolled growth of pathogens in aquatic organisms
and indiscriminate use of antibiotics to prevent them
have resulted in the emergence of several resistant
pathogens in aquaculture. Presently, these two factors
are the most important concerns for both researchers
and farmers. Diseases caused by infectious microor-
ganisms are known to be one of the major constraint in
the aquaculture industry for past many years (Scholz
et al. 1999) and are impeding the development and
sustainability of the industry throughout the world
(Bondad-Reantaso et al. 2005). Thus, there is a need to
devise suitable tools to control the disease outbreaks in
this sector. The concept of functional feed is an
emerging paradigm in aquaculture industry to develop
diets of balanced nutrition supplemented with feed
additives for improving the health and disease resis-
tance of cultured fishes (Gatlin and Li 2004). There is an
increased concern over the usage of antibiotics,
although some reports have shown that antibiotics
may improve growth and feed efficiency by killing the
microflora of intestine, thus resulting in enhanced
utilization of amino acid by the host organism (Rawles
et al. 1997). Use of antibiotics poses considerable
threats to the aquatic microorganisms prone to
development of antibiotic resistance and accumulation
of antibiotic residue from animals of lower food chain
to higher one including humans (FAO 2002). These
concerns prompted the ban on use of such therapeutics
in Europe, USA (Patterson and Burkholder 2003),
paving the way in searching for new avenues and
alternatives to replace antibiotic use against disease
outbreak. Alternative strategies such as use of vaccine,
dietary supplement of probiotics, prebiotics, and immu-
nostimulant may help to reduce the susceptibility of fish
to diseases. Immunostimulants are the modern and
primary tools in aquaculture that help in enhancing
resistance against infectious diseases by enhancing
innate humoral and cellular defense mechanisms.
Various kinds of substances have been used and their
suitability as immunostimulant has been studied, but
only few of them are found suitable for use in
aquaculture (Raa et al.1992; Siwicki et al. 1998). In
recent years, many review articles have been published
explaining different aspects of relationship between
immunostimulants and innate immunity system in fish
(Galeotti 1998; Gannam and Schrock 1999). Sohn et al.
(2000) discussed the role of immunostimulants in
monogastric animals and fishes in details. Efficiencies
and dose–effect relationship of immunostimulants in
marine fishes have been reviewed by Galindo-Villegas
and Hosokawa (2004). Immunostimulants and their
biological effects pertaining to fish larval aquaculture
were explained by Bricknell and Dalmo (2005),
whereas Magnado
ttir (2006) gave an overview of the
ontogenic development of non-specific immune system
with respect to different innate factors influenced by
immunostimulants. Later on, Sahoo (2007) and Mag-
ttir (2010) described immunological control of fish
diseases. More recently, immunological control of fish
diseases was discussed by Ringø et al. (2012), in which
the authors have discussed in detail about the inherent
and external factors affecting fish diseases. Use of
prebiotics is limited in aquaculture, which is gaining
importance slowly along with immunostimulants.
Ganguly et al. (2010) summarized the use of prebiotics
and probiotics as immunostimulants in aquaculture.
Various studies in fishes have proven b-glucan as a
potent, valuable, and promising immunostimulant for
improving immune status and controlling diseases in
fish culture (Robertsen et al. 1994; deBaulny et al.
1996; Anderson 1997; Figueras et al. 1998; Kawakami
et al. 1998; Robertsen 1999). The immunomodulatory
properties of b-glucans were first demonstrated in
432 Fish Physiol Biochem (2013) 39:431–457
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mammals wherein induced haemopoiesis and
enhanced immunity were observed resulting in an
increased resistance to infectious pathogens (Di Luzio
1985). Volman et al. (2008) outlined a broader picture
of immunomodulating effects of b-glucans in animals
and humans. Soltanian et al. (2009) summarized
b-glucan application in vertebrates (mice, guinea pig,
rat, pig, sheep, cattle, fish and rabbit) and invertebrates
(shrimp and artemia). In recent years, attention has
been focused on the use of b-glucan in fishes. Many
studies have been carried out on different fish species
such as Atlantic salmon (Paulsen et al. 2001), rainbow
trout (Jorgensen et al. 1993; Djordievic et al. 2009),
Snapper (Cook et al. 2003), African catfish (Yoshida
et al. 1995), Prawn (Hai and Fotedar 2009), and Sea
bass (Bagni et al. 2005, 2008; Bonaldo et al. 2007).
These studies have shown the effect of b-glucan on the
growth (Cook et al. 2003; Misra et al. 2006b), survival,
resistance, and protection against pathogen (Welker
et al. 2007; Sealey et al. 2008), antibody production
(Selvaraj et al. 2005; Kamilya et al. 2006), immune-
related gene expression (Løvoll et al. 2007; Zhang
et al. 2009), and as adjuvant (Rørstad et al. 1993;
Kawakami et al. 1998) in wide range of fish species.
Considering the multifaceted role of b-glucans in fish
immune system, a comprehensive and updated review
of effect of various sources of b-glucan through
different routes of administration, alone or in combi-
nation with other immunostimulants pertaining to fish
and shellfishes of both freshwater and marine origin, is
presented here. This review provides an in-depth
discussion on various roles played by b-glucan in fish
and shellfish immunology. Use of b-glucan as immu-
nostimulant in aquaculture (fish and shellfishes) has
been summarized in Table 1.
Types, sources, and structure of b-glucans
b-Glucans are naturally occurring polysaccharides
with glucose as structural component, linked by
b-glycosidic bonds. In nature, b
-glucans are wide spread
in the cell wall of many plants (wheat, rye, barley, and
oat), baker’s and brewer’s yeast (Saccharomyces
genus), and Echinaceae members (Tokunaka et al.
2000). Other sources of b-glucan include seaweed like
Laminaria sp. (Teas 1983), various species of mush-
rooms such as Shiitake (Lentinus edodes), Maitake
(Grifola frondosa), Reishi (Ganoderma lucidum)
(Wasser and Weis 1999), Schizophylan (Schizophyl-
lum commune), and SSG (Sclerotinia sclerotiorum)
(Brochers et al. 1999), and certain fungi (Agaricus
subrufesuns). b-glucans are also the structural con-
stituents of some of the pathogenic fungi, Pneumo-
cystis carini (Lebron et al. 2003), Cryptococcus
neoformans (Reese et al. 2007), and some bacteria
belonging to Rhizobiaceae family (Breedveld and
Milleri 1994). The common sources of b-glucan are
derived from the cell wall of baker’s yeast Saccharo-
myces cerevisiae and the most important among all are
b-1, 3 and 1, 6 glucan. Glucans are heterogeneous
group of glucose polymers, consisting of a backbone
of b (1, 3)-linked b-
D-glucopyranosyl units with b-(1,
6)-linked side chains of varying distribution and
length. b-glucans derived from different sources have
differences in their structure. Oat and barley b-glucans
are linear with b (1, 4) and (1, 3) linkages. Mushrooms
b-glucans have short b (1, 6)-linked branches from b
(1, 3) backbone. Yeast b-glucans have b (1, 6)
branches further with additional b (1, 3) regions.
These structural differences can trigger difficulties in
extraction and differences on their activity. Larger
molecular weight glucans activate leukocytes, stimu-
lating their phagocytic, cytotoxic, and antimicrobial
activities, and production of reactive oxygen species
(ROS). Low molecular weight glucans have less
cellular effects, whereas very short glucans are
considered as inactive (Akramiene et al. 2007).
Studies have shown that insoluble (1, 3/1, 6)
b-glucans have greater biological activity than that
of its soluble (1, 3/1, 4) counterparts (Ooi and Liu 2000).
Basically, glucan molecules are of two types on
the basis of glycosidic bonds present in them, that is,
a-glucan (dextran with 1,6, starch with a-1,4- and
a-1,6-glycosidic bonds) and b-glucan (cellulose with
b-1,4, zymosan with b-1,3, laminarin with b-1,3- and
b-1,6, lichenin with b-1,3 and b-1,4 glycosidic
bond). Because of complex structure in b-glucans,
they have superior ability to activate the immune
response and act as biological response modifiers
(BRM) (Miura et al. 1996). Certain characteristics of
this glucan such as ability to function normally on
immune system without over activating them (Chi-
hara 1992), ability to lower the elevated levels of
cholesterol (Behall et al. 1997; Bell et al. 1999;
Braaten et al. 1994), and ability to reduce sugar
levels (Wood 1990; Pick et al. 1996) make it unique
among immunostimulants.
Fish Physiol Biochem (2013) 39:431–457 433
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Table 1 Use of different b-glucan in aquaculture
Species Glucan References
1. Nile tilapia (Oreochromis niloticus) Yeast and yeast subcomponent Shelby et al. (2009)
2. Carp (Labeo rohita) Yeast cell wall Pal et al. (2007)
3. Pacific white shrimp (Litopenaeus vannamei) Inactive yeast cell wall Chotikachinda et al. (2008)
4. Gilthead seabream (Sparus aurata L.) Whole yeast cell Cuesta et al. (2007)
5. Black tiger shrimp (Penaeus monodon) Brewers yeast (b-glucan) Suphantharika et al. (2003)
6. Channel catfish (Ictalurus punctatus) YYS/whole cell Welker et al. (2007)
7. Black tiger shrimp (Penaeus monodon) Crudlan,zymosan(b-1,3-glucan) Sritunyalucksana et al. (1999)
8. Red tail Black shark (Epalzeorhynchos
MacroGard ? Aquagen Russo et al. (2006)
9. Pacific white shrimp (Litopenaeus vannamei) b-1,3-Glucan ? ascorbic acid Lopez et al. (2003)
10. Tiger shrimp (Penaeus monodon) b-Glucan ?Vibrio bacterian Pais et al. (2008)
11. Large yellow croaker (Pseudosciaena
b-1,3-Glucan Ai et al. (2007)
12. European sea bass (Dicentrarchus labrax) b-1,3,1,6-Glucan Bonaldo et al. (2007)
13. Fathead minnows (Pimephales promelas) b-1,3,1,6-Glucan Palic et al. (2006)
14. Atlantic salmon (Salmo salar L) b-1,3,1,6-Glucan (yeast) Rørstad et al. (
15. Rainbow trout (Oncorhynchus mykiss) b-1,3,1,6-Glucan (yeast) Verlhac et al. (1998)
16. Atlantic cod (Gadus morhua L.) b-1,3,1,6-Glucan Skjermo et al. (2006)
17. White shrimp (Litopenaeus vannamei) b-1,3-Glucan (S. commune) Wang et al. (2008)
18. Black tiger shrimp (Penaeus monodon) Carboxymethyl b-1,3-glucan Klannukarn et al. (2004)
19. Black tiger shrimp (Penaeus monodon) b-1,3-Glucan (laminarin) Sritunyalucksana et al. (2002)
20. Black tiger shrimp (Penaeus monodon) b-1,3-Glucan (brewers yeast) Thanardkit et al. (2002)
21. White shrimp (Penaeus vannamei) b-1,3-Glucan (yeast) Scholz et al. (1999)
22. Kuruma shrimp (Penaeus japonicus) b-1,3-Glucan (yeast) Namikoshi et al. (2004)
23. Kuruma shrimp (Penaeus japonicus) b-1,3-Glucan (S. commune) Itoh (1997)
24. Black tiger shrimp (Penaeus monodon) b-1,3-Glucan (S.commune) Chang et al. (1999, 2000, 2003)
25. Rohu (Labeo rohita) b-1,3-Glucan Sahoo and Mukherjee (2001,
26. Asian catfish (Clarias batrachus) b-1,3-Glucan (yeast) Kumari and Sahoo (2006a, b)
27. Carp (
Cyprinus carpio) b-1,3-Glucan (yeast) Selvaraj et al. (2005)
28. Rainbow trout (Oncorhynchus mykiss) b-1,3-Glucan (laminaran) Løvoll et al. (2007)
29. Carp (Cyprinus carpio) b-1,3-Glucan ? LPS Selvaraj et al. (2006)
30. Indian white shrimp (Fenneropenaeus
Alkali soluble and insoluble b-1,3-
Anas et al. (2009)
31. Carp (Cyprinus carpio) b-Glucan (yeast) Selvaraj et al. (2005)
32. Zebrafish (Danio rerio) b-Glucan(S. cerviciaea) Rodrı
guez et al. (2009)
33. Rainbow trout (Oncorhynchus mykiss) b-Glucan (barley) Sealey et al. (2008)
34. Sea bass (Dicentrarchus labrax) Yeast b-glucan (Macrogard) Bagni et al. (2005)
35. Nile tailapia (Oreochromis niloticus) b-Glucan Whittington et al. (2005)
36. Pink snapper (Pagrus auratus) Ecoactiva Cook et al. (2003)
37. Catla (Catla catla) Mushroom glucan Kamilya et al. (2006)
38. Indian white shrimp (Fenneropenaeus
Marine yeast glucan (Candida sake
Sajeevan et al. (2009)
39. Black tiger shrimp (Penaeus monodon) Bakers yeast (S. cerviciae) Huang and Song (1999)
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Mechanism of action of beta-1,3/1,6-glucan
b-Glucan plays important role in the activation of both
innate and acquired immune functions. Innate immune
responses stimulated by b-glucans not only act on
invading microorganisms but also complement the
activation and action of acquired immunity (Sakai
1999). b-glucans are responsible for a multitude of
actions which protect and enhance the immune system
and provide optimum resistance to any possible health
assailants due to its ability to bind directly with
macrophages and other white blood cells (neutrophils
and natural killer (NK) cells) and to activate them
(Gantner et al. 2003; Herre et al. 2004). Macrophage
cells are one of the principal cell types involved in
natural immunity. When b-glucan receptors are engaged
by beta 1,3/1,6 glucans, all immune functions are
improved, including phagocytosis (ability to engulf
foreign cells and particles), release of certain cytokines
(intercellular hormones) IL-1, IL-6, GM-CSF, interfer-
ons, and the processing of antigens. These cytokines
stimulate formation of new white blood cells (WBC)
thus providing immunity to b-glucan binding receptors
present in all vertebrates ranging from fish to human.
Fishes have both specific and non-specific defense
mechanism. The activated phagocytic cells and WBC (B
and T cells) produce cytokines and antibodies, respec-
tively, and enhance the efficacy of vaccines (Raa 2000).
Crustacean immune systems have the ability to detect
foreign components such as LPS and b-glucan present in
the cell wall of microorganisms. In shrimp, specific
binding proteins to carbohydrate moieties are present in
serum as recognition proteins that activate the cellular
functions when they react with the microbial LPS or
b-glucan (Vargas-Albores and Yepiz-Plascencia 2000).
These microbial components activate the functions of
encapsulation, coagulation, melanization, and phagocy-
tosis associated with defense mechanism. b-glucan
binding proteins interact with b-glucan molecule and
form a complex of BGBP-BG that reacts with the
hemocyte surface and releases the hemocytic granules
which in turn leads to the activation of proPOs (Vargas-
Albores et al. 1996). Johansson et al. (1999)havecloned
and characterized the cellular receptors present on the
surface of hemocyte. The hemocyte granule plays
important role in storing the proPOs enzyme and its
release by exocytosis. The enzyme promotes the oxidation
of phenol to o-phenols to quinines, which have the ability
to kill the infectious pathogens (Soderhall et al. 1994).
b-Glucan receptors
Induction of cellular response by b-glucan is associ-
ated with specific interactions of one or more cell
surface receptors. Glucans are believed to modulate
innate immunity by binding to specific receptors on
monocyte/macrophages, neutrophils, and natural
killer (NK) cells (Muller et al. 2000). Different
b-glucans are associated with different or same type
of receptors. b-glucan receptor was first identified as
opsonin-independent receptor for the activation of
alternative complement activation pathway (Czop and
Austen 1985). They bind to pattern recognition
receptors and modulate innate immunity by activating
the macrophage cells. Available reports suggest that
there are multiple glucan binding sites on macro-
phages but the nature of the glucan receptors and exact
mechanism for modulating innate host defenses are
not clear. Progress made to understand the process of
interactions of glucan and its receptors includes
binding of type 3 complement receptor (CR3) to
glucans by Vetvicka et al. (1996), report of lactosyl-
ceramide a glucan binding moiety on immunocytes by
Zimmerman et al. (1998), existence of non-CR3
glucan binding (Dectin-1) sites on human monocyte/
macrophages and human dermal fibroblasts by Rice
et al. (2002), reports on a water-soluble, polyanionic,
carboxymethylated (CM) glucan binding to mouse
peritoneal macrophages via scavenger receptors (SRs)
by Vereschagin et al. (1998), reports of Drosophila SR
binding to laminarin, a low molecular weight glucan
and other microbial cell wall components by Pearson
et al. (1995).
Important distinction between innate and adap-
tive immunity is the difference in their receptors
that are responsible for immune recognition (Bende-
lac and Fearon 2000; Rice et al. 2002). Innate
immunity is the first line of defense against
microbial infection, where genetically pre-deter-
mined pattern recognition receptors (PRRs) recog-
nize bio-molecules (carbohydrates, lipids, and
proteins) present in the cell wall of microorganisms.
This phenomenon is called as pathogen-associated
molecular patterns (PAMPs). However, adaptive
immunity requires somatically generated receptors
that recognize antigenic patterns to which the host
has been previously exposed (Peiser and Gordon
2001; Rice et al. 2002). Different types of b-glucan
receptors are as follows:
Fish Physiol Biochem (2013) 39:431–457 435
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Scavenger receptor
Based on structural characteristics, scavenger recep-
tors are divided into three classes: SR-A, SR-B, and
SR-C. Most of the SRs are non-opsonic receptors that
display low-affinity and bind to many polyanionic and
modified substances. They may bind to anionic
b-glucans (sulphated b-glucans either made chemi-
cally or found in certain algae), but does not involve in
b-glucan binding and internalization (Dennehy and
Brown 2007).
Complement receptor
Complement receptor (CR3) belongs to the family
-integrin, a heterodimeric transmembrane glycopro-
tein, which consists of CD11b and a non-covalently
associated CD18. These receptors are highly
expressed on neutrophils, monocytes, and NK cells
compared to macrophages (Thornton et al. 1996a).
The CD11b subunit of CR3 has a functional domain A
or I. These domains are important for phagocytosis
and binding of iC3b-coated particles, while the other
lectin domain located C-terminal to the I-domain is
responsible for the non-opsonic binding properties of
CR3. The leukocyte b
-integrin also known as Mac-1,
complement receptor type 3 (CR3), and CD11b/CD18
function both as adhesion molecule facilitating diape-
desis and as CR3 enabling phagocytosis or degranu-
lation in response to factor I-cleaved C3b fragment of
C3 (iC3b)-opsonized microorganisms. The same lec-
tin domain within CD11b regulates both the cytotoxic
and adhesion functions of Mac-1/CR3. CR3 (Mac-1,
CD11b/CD18, or µMb2-integrin) has been identified
as the leukocyte membrane receptor for b-glucans
(Thornton et al. 1996b). The role for CR3 in b-glucan-
induced tumoricidal activity was also investigated by
Vetvicka et al. (1996) who have reported that the
soluble CR3-specific polysaccharides such as b-glu-
can induces a primed state of CR3 which could trigger
killing of iC3b-target cells that were otherwise resis-
tant to cytotoxicity.
Lactosylceramide (LacCer; CDw17) is a glycosphin-
golipid found in the plasma membranes of many cells.
Interaction of b-glucan with lactosylceramide receptor
can induce macrophage inflammatory protein (MIP)-2
and activate NFkB, which can enhance the neutrophil
oxidative burst and antimicrobial functions. It has
been identified as a b-glucan receptor from biochem-
ical analyses of the interactions between b-glucan and
isolated human leukocyte membrane components
(Zimmerman et al. 1998). It was found that lactosyl-
ceramide may bind specifically to b-glucan with
concomitant production of reactive oxygen metabo-
lites (Chen and Seviour 2007).
Dectin-1 (b-glucan receptor)
Dectin-1 (bGR) is a non-classical C-type lectin that
predominantly binds protein ligands and is the primary
PRR for glucans (Brown et al. 2003). bGRs have been
considered to be the major b-glucan receptor (Den-
nehy and Brown 2007). It consists of single C-type,
lectin like, carbohydrate recognition domain with a
short stalk, and a cytoplasmic tail possessing an
immunoreceptor tyrosine-based activation motif. It
recognizes carbohydrates containing b-1,3 and/or
b-1,6 glucan linkages and is expressed on cells of
the monocyte/macrophages and neutrophils. Dendrite
cells and a subpopulation of T cells are also known to
express the bGR, but at a lower level (Taylor et al.
2002). Dectin-1, in association with TLR2, resulted in
the activation of a macrophage’s proinflammatory
response to mycobacterial infection (Yadav and
Schorey 2006). Structural relationship of glucan and
Dectin-1 has been studied by Adam et al. (2008). They
have reported that Dectin-1 is highly specific for
glucans that have a (1 ? 3)-b-
Toll-like B-glucan receptors
The innate immune system is an evolutionally con-
served host defense mechanism initiated by pattern
recognition receptors (PRRs). Among many PRRs,
Toll-like receptors (TLRs) are capable of sensing
organisms ranging from bacteria, fungi, protozoa to
viruses. TLRs recognize pathogens either on the cell
surface or in the lysosome/endosome compartment.
Recently, cytoplasmic PRRs have been identified to
detect pathogens that have invaded cytosols (Uematsu
and Akira 2006). These type I transmembrane recep-
tors identify microbial conserved structures or path-
ogen-associated molecular patterns (PAMPs).
Recognition of microbial components by TLRs
436 Fish Physiol Biochem (2013) 39:431–457
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initiates signaling transduction pathways and induces
gene expression. These gene products regulate innate
immune responses and further develop as an antigen-
specific acquired immunity. TLR signaling pathways
are regulated by intracellular adaptor molecules, such
as MyD88, TIRAP/Mal, and provide specificity of
individual TLR-mediated signaling pathways. TLR-
mediated activation of innate immunity is involved not
only in host defense against pathogens but also in the
immune disorders (Arancibia et al. 2007). The cyto-
plasmic portion of a TLR is similar to that of the
interleukin (IL)-1 receptor family. It is therefore called
the Toll/IL-1 receptor (TIR) domain. A TIR domain is
required for initiating intracellular signaling. More-
over, the extracellular region of TLRs and IL-1R are
markedly different. However, IL-1R possesses an Ig-
like domain where as TLRs contain LRRs in the
extracellular domain. LRRs are responsible for the
recognition of PAMPs (Akira et al. 2006). Fungal cells
and zymosan consist of many different molecular
moieties that bind to TLR2 and TLR4 facilitating
myD88-dependent intracellular signaling that induce
cytokines favoring Th1 cell differentiation (Romagne
Molecular characterization of BGR, BGBP, proPO
in fish
b-Glucan receptors (BGR)
Toll-like receptor 4 (TLR4) is critical for the recog-
nition of LPS, cellular responses, and some viral
envelope proteins. Expression of zebra fish TLR4,
TLR2, and MyD88 gene was reported in the adult stage
and was expressed at higher levels in fish infected with
the pathogen Mycobacterium marinum (Meijer et al.
2004). The same gene from rare minnow, Gobiocypris
rarus (GrTLR4b) expressed its mRNA in gill, heart,
intestine, kidney, liver, muscle, and spleen tissues and
was up-regulated after challenging with grass carp
reovirus or Aeromonas hydrophila (Su et al. 2009a).
TLR3 mRNA transcripts from grass carp (ciTLR3)
were significantly up-regulated following GCRV
infection (Su et al. 2009b) and its polypeptide
sequences of subunits b2 (CD18) and aM (CD11b)
in rainbow trout exhibited similarity with human,
mouse, and zebrafish orthologs. The main source of
the trout CD18 and CD11b-like mRNA transcripts was
kidney (Mikrou et al. 2009). TLR9 family sense viral
and bacterial DNA present in the endosomal compart-
ment. The full-length TLR9 cDNA was cloned from
flatfish species, half-smooth tongue sole (Cynoglossus
semilaevis) (CsTLR9) which was expressed in spleen
and gonads and appeared to be developmentally
regulated (Yu et al. 2009). The same receptor
(TLR9) was cloned from Gilthead sea bream (Sparus
aurata L.) and expressed in immune-related organs
(spleen, head kidney) and mucosal–epithelial barriers
(gills, gut, and skin) where infection with pathogen
showed no difference in TLR9 expression compared
with control (Franch et al. 2006). Japanese flounder,
Paralichthys olivaceus TLR 9 cDNA highly expressed
in epithelial and lymphoid organs. The mRNA copy
number of TLR9 and its adapter protein, MYD88,
were enhanced in blood, gill, kidney, and spleen after
challenging with Edwardsiella tarda and expressed in
kidney cells (Takano et al. 2007). TLR9 gene from
Atlantic salmon (Salmo salar) showed elevated
expression in head kidney leukocytes after in vitro
treatment with CpG ODNs and recombinant trout
interferon (IFN)-c (Skjæveland et al. 2008).
The full-length TLR from goldfish (Stafford et al.
2003), constitutively expressed in macrophages,
spleen, and kidney. Bacterial LPS, heat-killed Aero-
monas salmonicida, and live Mycobacterium chelonae
significantly up-regulate the TLR mRNA expression.
Japanese flounder, JF-TLR2 and JF-TLR22 gene are
homologs to human TLR2 and fugu TLR22 and mainly
expressed in peripheral blood leukocytes (PBLs),
kidney, spleen, and gill. Their expression was induced
by both peptidoglycan and polyI:C, but number of JF-
TLR-expressing cells did not change (Hirono et al.
2004). Atlantic salmon (Salmo salar) cloned SsTLR8
mRNA expression was reported as tissue-specific,
with highest level in the spleen, and SsMyD88 in all
the tested tissues. Their expression was up-regulated
into cells treated with recombinant IFN a1 and IFN g.
Expression of SsTLR8 had no effect following
challenge with salmon alpha virus subtype 3 (SAV3)
in vivo (Skjæveland et al. 2009). Full-length cDNA
and gene sequences of TLR5S, TLR20, and TLR21
from catfish were reported to be single copy genes in
catfish (Baoprasertkul et al. 2007).
Toll receptor gene (MjToll) from kuruma shrimp,
Marsupenaeus japonicus, constitutively expressed in
the gill, gut, lymphoid organ, heart, hematopoietic
organ, hemocyte, ventral abdominal nerve cord,
Fish Physiol Biochem (2013) 39:431–457 437
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eyestalk neural ganglia, and brain tissues. Peptidogly-
can enhanced the expression (76-fold) compared to
control in lymphoid organ, in vitro (Mekata et al.
2008). Partial Toll receptor gene of giant tiger shrimp,
Penaeus monodon, expressed in gut, gill, and hepato-
pancreas on challenging with WSSV and showed
clustering with Toll1 and Toll5 gene products (Arts
et al. 2007).
Lipopolysaccharide and b-glucan binding protein
Pattern recognition proteins (PRPs) recognize com-
mon epitopes present on the surface of invading
pathogen and are generally carbohydrate moieties
(Fearon and Locksley 1996). Invertebrate protein
recognizing carbohydrate from microbial surface has
been isolated and showed their involvement in
immunity (Sritunyalucksana and Soderhall 2000). A
number of PRPs has been isolated and characterized.
LGBP gene was cloned from Fenneropenaeus chin-
ensis, Fc-LGBP, and its expression was down-regu-
lated 24 h post-injection of bacteria. Bacterial binding
assays showed strong binding activity of LGBP to
Gram-negative bacteria (Du et al. 2007). The same
was expressed only in the hemocyte and hepatopan-
creas of Chinese shrimp Fenneropenaeus chinensis
and transcription of LGBP was enhanced in response
to bacterial infection (Liu et al. 2009). The Chinese
mitten crab Ericdheir sinensis, LGBP gene (Es-
LGBP) was cloned and highly expressed in hemocytes
and the lowest in the stomach. Es-LGBP gene
expression was up-regulated on binding to LPS and
b-1,3-glucan and bacterial infection (Zhao et al. 2009).
The same gene transcript in the hemocyte of white
shrimp Litopenaeus vannamei was increased in 3 and
6 h of post-Vibrio alginolyticus injection (Cheng et al.
2005). Disk abalone (Haliotis discus discus), Pattern
recognition protein (HD-PRP) was expressed in gill,
mantle, digestive tract, hepatopancreas and hemo-
cytes, and the expression was enhanced with the
administration of Vibrio alginolyticus, LPS, b-1,3-
glucan (Nikapitiya et al. 2008). A high-density
lipoprotein, b-glucan binding protein (bGBP-HDL)
cloned from white shrimp Litopenaeus vannamei
showed its expression in hepatopancreas, muscle,
pleopods, and gills and involved in immune response
(Romo-Figuero et al. 2004). Black tiger shrimp
Penaeus monodon b-1,3-glucan binding protein
(GBP) expressed in hemocyte and showed no signif-
icant change in mRNA expression of shrimps injected
with curdlan or heat-killed bacterial cell of Vibrio
harveyi within 12 h post-injection (Sritunyalucksana
et al. 2002).
Prophenoloxidase (proPO)
Crustaceans lack normal defense mechanism as it
occurs in fishes, but involves proPO, which is one of
the main defenses functioning as a non-self recogni-
tion system. This system is triggered by lipopolysac-
charides (LPS) or peptidoglycans (PGN) from bacteria
and b-1, 3-glucans from fungi. There are proteins
involved in the recognition of LPS, peptidoglycans,
and b-1, 3-glucan, which have been named as pattern
recognition proteins (PRPs) and are involved in
various ways in the humoral defense mechanisms in
crustaceans. The proPO activation pathway, like the
vertebrate complement system, is a proteolytic cas-
cade containing several serine proteases and their
inhibitors. It is known that one of the serine protease in
the cascade, named as proPO-activating enzyme
(ppA), will cleave proPO to generate the active
enzyme, phenoloxidase (PO). This enzyme can pro-
duce toxic compounds to microorganisms by oxidiz-
ing phenols to melanin.
In crustaceans, several proteins of this enzyme
cascade have been isolated and partially characterized
(Soderhall et al. 1990). ProPO, the latent inactive pro-
form of phenoloxidase, has been purified from crayfish
blood and found to possess a molecular mass of
76 kDa (Aspan and Soderhall 1991). It is converted
into its active form by a serine protease, ppA, and the
resulting active forms of phenoloxidase has molecular
masses of 62 and 60 kDa (Aspan and Soderhall 1991).
The proPO activation enzyme, ppA, has also been
purified and characterized of having molecular mass
of 36 kDa (Aspan et al. 1990). These proPO system
proteins are contained in the blood cells of crayfish
(Soderhall 1981) and can be released by a regulated
exocytosis (Johansson and Soderhall 1989).
Phenoloxidase (PO) is the terminal enzyme of the
whole cascade system. Once generated, it oxidizes
dihydroxy phenylalanine (DOPA) to dopaquinone,
which is converted to melanin (a brown pigment)
through several non-enzymatic steps (Soderhall 1982;
Sritunyalucksana and Soderhall 2000). A direct anti-
microbial activity has been described for melanin and
438 Fish Physiol Biochem (2013) 39:431–457
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its precursors (Nappi and Vass 1993). Besides, the
production of reactive oxygen species such as super
oxide anions and hydroxyl radicals during the gener-
ation of quinoids (Song and Hsieh 1994; Nappi et al.
1995) also has an antimicrobial role. The generated
PO plays an important role in invertebrate defense, as
it can melanize pathogens (Soderhall and Cerenius
1998), sclerotize the cuticle (Sugumaran 1991), and
heal wounds (Lai-Fook 1966). In addition, PO also
performs other biological activities such as mediating
the cell adhesion of crayfish blood cells (Johansson
and Soderhall 1988) and promoting encapsulation
(Kobayashi and Soderhall 1990).
The activity of PO should be tightly regulated to
avoid melanin formation at sites other than those of
infection. Proteinase inhibitors are present in plasma
such as a trypsin inhibitor, with a mass of 155 kDa
(Hergenhahn et al. 1987) and a
macroglobulin (Hall
et al. 1989). Both these high molecular mass protein-
ase inhibitors can inhibit ppA and thus inhibiting the
activation of the proPO system, with the trypsin
inhibitor being more efficient (Aspan et al. 1990).
Besides several proteins of this cascade, proPO
gene from several species has been cloned and
characterized and demonstrated specific expression.
Similarly, expression varied with life stages of
animals, health status, and environmental conditions.
In white shrimp (L. vannamei), proPO gene was
cloned and its mRNA expression was confirmed in
hemocyte, but not in the hepatopancreas or muscle
(Lai et al. 2005). Similarly, proPo gene in giant fresh
water prawn (Macrobrachium rosenbergii) was
observed in hemocyte only and its transcript expres-
sion was observed highest at B stage and lowest at D2/
D3 stage (Liu et al. 2006), and proPO mRNA showed
highest level of expression in vivo, with 5 lg of CpG
oligodeoxynucleotide injection (Lu et al. 2006). Red
swamp cayfish (Procambarus clarkii) proPO sequence
showed similarity with arthropod proPO and it was
closely related with freshwater crayfish P. leniusculus
(Li et al. 2009). All the proPOs sequence cloned
illustrated some common regions. Mud crab (Scylla
serrata) proPO gene showed strongest expression in
hemocyte and its level of expression was significantly
increased within 12–14 h of post-injection with LPS
(Ko et al. 2007).
A new proPO was cloned from white shrimp
Litopenaeus vannamei (Ai et al. 2008, 2009; Yeh et al.
2009). proPO-b was detected mainly in hemocyte as
well as in gill, midgut, heart, stomach, posterior
midgut cecum, and cuticular epidermis. Sequence of
both proPO-a and proPO-b showed microsatellite site
at the 3’ end of open reading frame (ORF) (Ai et al.
2008). Similarly, LvproPO-2 showed similarity with
LvproPO-1 of white shrimp and expression was
observed on the surface and nucleus of the hemocytes,
but not in plasma (Ai et al. 2009). ProPO-I (cloned
early) and proPO-II mRNAs of shrimp are expressed
in hemocytes and located on different loci. Shrimps
fed with diet containing sodium alginate alone or in
combination with Vibrio alginolyticus showed mRNA
expression of both proPOs at different molt stages
(Yeh et al. 2009).
b-Glucan on protein and gene expression
Role and potential influence of b-glucan on immune-
related gene and protein expression in different fish
species have been reported by many authors. Head
kidney macrophages cells from rainbow trout (On-
corhynchus mykiss) and Atlantic salmon immunosti-
mulated with LPS and b-glucan showed enhanced
levels of ILs, but transcription of C3 was not induced
in both trout and salmon. Complement C3 subtypes
were differentially regulated after 48 h in vivo stim-
ulation with LPS and b-glucan. These results sup-
ported the earlier findings of absence of C3 in
macrophages of the spotted wolf fish (Anarhichas
minor) (Løvoll et al. 2007). Similarly, oral adminis-
trated b
-1, 3-glucan in tilapia (Oreochromis niloticus)
for 5 days stimulated production of cytokine-like
proteins of tumor necrosis factor-a (TNF-a), IL
(interleukin)-1b, IL-10, IL-12 in fish plasma (Chansue
et al. 2000). Many authors have reported the presence
of IL-Ib (Secombes et al. 1996; Ellsaesser and Clem
1994; Verburg van Kemenade et al. 1995) and TNF-a
(Zelinkoff et al. 1990; Ahne 1993) in fishes. However,
Hardie et al. (1994) have reported specific TNF-a
receptor in rainbow trout lymphocytes. b-glucan
administered through bath treatment in rainbow trout
fry at two different doses (0.1 and 1.0 lM) for 45 min,
four times with an interval of 1 week, showed
enhanced gene expression with regard to the pro-
inflammatory cytokines IL-1b, TNF-a, IL-6, the anti-
inflammatory cytokines IL-10 and TGF-b at first bath.
Fish Physiol Biochem (2013) 39:431–457 439
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However, no significant change in the pro-inflamma-
tory cytokine IL-17A transcripts, compared to control,
was observed. Gene expression levels in b-glucan
treated fish after the fourth bathing showed no
significant differences compared to control (Zhang
et al. 2009). The same were fed with lentinan, a
mushroom b-glucan, for 37 days and administered
with bacterial LPS, an inflammation inducing agent.
Use of lentinan showed decrease in the expression of
genes involved in acute inflammatory reaction to
inflammatory agents compared to fish fed with bac-
terial LPS, but no change in expression of gene
associated with major immune response was observed
(Djordievic et al. 2009).
Grass carp treated with b-glucan for 15 days prior
to grass carp hemorrhage virus (GCHV) injection
showed increased MX gene expression levels during
early stages (12 and 36 h) of GCHV infection and
significantly improved the survival rate to 60 %.
Increased superoxide dismutase (SOD) and catalase
(CAT) activities of erythrocytes and Mx gene expres-
sion were observed compared to the group not pre-
treated with b-glucan, indicating that b-glucan
enhances anti-viral responses (Kim et al. 2009). No
significant response on the expression of TNF-a or IL-
1b was reported in fish fed with 5 mg/ml of b-glucan
(S. cerevisiae), but modulation of IFN-c and chemo-
kine expression in kidney were reported (Rodrı
et al. 2009).
Feeding Pacific white shrimp with different dos-
ages viz. 0, 0.2, 1.0 % of b-glucan (Schizophyllum
commune) for 1 week showed up- and down-regula-
tions of gene within 24 h. Penaeidin 3 (Litvan PEN3)
was down-regulated (0–24 h), b-glucan binding pro-
tein–high density lipoprotein (BGBP-HDL) and lipo-
polysaccharide/b-glucan binding protein (LGBP)
showed a delayed up-regulation (3–7 days), but
hemocyanin, crustin, prophenoloxidase (proPO), and
transglutaminase (TGase) showed no response. Diet
with 2 g glucan/kg feed was sufficient for gene
expression (Wang et al. 2008). Gene expression in
Pacific white shrimp was observed to be site specific
such as BGBP-HDL, LGBP and hemocyanin are
expressed in hepatopancreas. Propo, TGase, penaeidin
3, crustins, and lysozyme showed highest expression
in hemocyte and lowest in hepatopancreas; moreover,
cMnSOD showed its highest expression in stomach
and muscle while lowest in hemocyte, midgut, neural
ganglion, and hepatopancreas (Wang et al. 2007a, b).
b-Glucan as immunostimulant
b-Glucan as immunostimulant has been widely stud-
ied in several vertebrate and invertebrate species and
discussed in detail by Soltanian et al. (2009). Dietary
administration of b-glucan to Atlantic salmon (Salmon
) showed stimulation of respiratory burst activity
(RBA) of head kidney macrophage in vitro. In vivo
experiment showed negative effect on RBA, serum
lysozyme production, and disease resistance to amoe-
bic gill disease (AGD) on challenging them with the
infectious pathogen (Bridle et al. 2005). A number of
reports reveal that dietary b-glucan administration
increases resistance to infection for short time periods
in Chinook salmon, Onchoryhnchus tshawytscha
(Nikl et al. 1993), African catfish, Clarius gariepinus
(Yoshida et al. 1995), gilthead sea bream, Sparus
aurata (Ortuno et al. 2002), and Indian carp, Labeo
rohita (Sahoo and Mukherjee 2002). Time of admin-
istration of b-glucan plays significant role in induction
of resistance against microsporidian, Loma salmonae,
in the rainbow trout (Guselle et al. 2007).
Bagni et al. (2005) reported duration-dependant
effect of dietary glucan where significant elevation of
serum complement activity in sea bass fed with alginic
acid and glucans at 15 days was reported; however,
serum lysozyme, gill and liver HSP concentrations
were enhanced at 30 days. Increased complement
activity was reported only in fishes fed with Ergosan
diet. Long-term period had no significant impact on
innate and specific immune parameters, survival,
growth performances, and conversion index in treated
and control fish. The quality of immunostimulant (b-
glucan) supplied plays crucial role in enhancing the
immune responses. Use of commercial b-glucan
products has showed their effects on immune
response. In an in vitro study, head kidney macro-
phage of pink snapper (Pagrus auratus) pre-incubated
with commercial b-glucan (EcoActiva
) and subse-
quently induced either by phorbol myristae acetate
(PMA) or lipopolysaccharide (LPS) resulted in sig-
nificant stimulation of superoxide anions and respira-
tory burst activity compared to induction of
macrophage with EcoActiva alone (Cook et al.
2001). Result of this study demonstrates that feeding
of b-glucan may enhance the recognition of LPS
present in the cell wall of Gram-negative fish patho-
genic bacteria resulting in improved killing efficiency
of macrophage to these pathogens. In another study,
440 Fish Physiol Biochem (2013) 39:431–457
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oral administration of EcoActiva in Pink snapper
increased macrophage O
radicals especially in win-
tertime only, but no enhancement in classical and
alternative pathway activity was seen, indicating
winter time to be the most favorable to feed snappers
for disease resistance (Cook et al. 2003). Glycans Bar,
krestin, scleroglucan, and zymosan in diets showed
significant increase in the survival rates of tilapia and
grass carp after injection with Aeromonas hydrophila
(Wang and Wang 1997). The effects of feeding 1,3/1,6
b-glucans on the innate and the adaptive immune
responses were studied in European sea bass (Dicen-
trarchus labrax) (Bonaldo et al. 2007).
Yeast b-1, 3/1, 6-glucan have been used for in vitro
and in vivo experiments to study degranulation of
primary granules in fish neutrophils (Palic et al. 2006).
b-glucan supplied to non-stress (NS), acute stress
(AS), and chronically stressed (CS) fish showed
increase degranulation in NS and prevented decrease
of degranulation in AS, whereas in CS fish, degran-
ulation reached to NS level after 3 days of feeding in
fathead minnows (Pimephales promelas, Rafins-
esque). Results indicated that b-glucan supplementa-
tion prior to AS and during CS in fish diet can enhance
neutrophils function and increase disease resistance
and survival rate. Immunomodulatory effect of dietary
b-1, 3/1, 6-yeast glucan showed enhanced effect on
concanavalin A, which induced proliferation of lym-
phocytes and antibody response after vaccination with
formalin-killed Yersinia ruckeri against red mouth
disease in rainbow trout. It had also showed enhanced
macrophage, lysozyme, and complement activity, but
no effect was observed on oxidative burst, pinocytosis,
and alternative complement pathway activation (Ver-
lhac et al. 1998). Wang et al. (2007a, b) used five
different glycans (Barley, Krestin, MacroGard, Scle-
roglucan and Zymosan) to study the immune response
in hybrid Tilapia and Japanese eels Anguilla japonica.
They showed increase lysozyme activity and phago-
cytic activity in both anterior kidney and peripheral
blood phagocytes. In vitro and in vivo conditions,
classical complement pathway (CCP), hemolytic
complement titer, and alternative pathway-hemolytic
complement titer improved in glucan treated tilapia.
Intramuscular injection of b-glucan (Saccharomyces
cerevisiae) in zebrafish (Danio rerio) on 6th day
before challenge with Aeromonas hydrophila showed
significant reduction in the mortality, increase in the
myelomonocytic cell population in the kidney, and
ability of kidney cells to kill A. hydrophila (Rodrı
et al. 2009). Protective effect of b-glucan injection
against several infections (Selvaraj et al. 2005; Misra
et al. 2006a; Anderson and Siwicki 1994) and dose-
dependent response to intramuscular injection with
b-glucan (Selvaraj et al. 2005
) have been demon-
strated in different species.
Similar studies have been done in invertebrates.
Indian white shrimp fed with marine yeast glucan once
in every 7 days showed significant survival against
WSSV (Sajeevan et al. 2009). However, increased
ProPO and reactive oxygen intermediate activity were
demonstrated with alkali insoluble glucan (AIG, rich
in (1 ? 3)-b-
D-glucan) from a filamentous fungi
Acremonium diospyri, whereas alkali soluble glucan
(ASG, rich in (1 ? 3)-a-glucan) did not show signif-
icant immune response. Their results revealed that
glucan can be used as a potential immunostimulant for
shrimp, where it should contain (1 ? 3)-b-
as the major fraction (Anas et al. 2009). Dietary
supplementation of b-1, 3/1, 6-glucan from Saccha-
romyces cerevisiae was used in spawners of Penaeus
monodon to study maternal transmission of immunity
to white spot syndrome caused by WSSV. Glucan fed
shrimp showed clinical symptoms of red body color-
ation and white spot on the shell, while larvae showed
protection against WSSV when challenged and
showed enhanced RPS of larva compared to the
control, clearly indicates maternal transmission of
immunity in invertebrates (Huang and Song 1999).
Soltanian et al. (2007a) challenged Artemia nauplii
with Vibrio campbellii under gnotobiotic conditions to
study anti-infectious potential of six commercial
b-glucans. Their results explained that the quality of
glucans (molecular weight, structure ratio of 1, 3/1, 6
glucan ratio and branching) is more important than the
quantity of the product offered in diet. Application of
commercial b-glucan sources together with chitin has
been reported to enhance disease resistance in Artemia
(Soltanian et al. 2007b).
Feeding b-glucans to clam (Ruditapes decussatus)
and Mediterranean mussel (Mytilus galloprovincialis)
showed improved immune response. Increased nitric
oxide production was observed in both the species but
release of free oxygen radicals and phorbol 12-myr-
istate 13-acetate (PMA) was enhanced in mussel
hemocytes. However, high dose of b-glucans com-
bined with zymosan decreased there respiratory burst
activity in mussel. Hemolymph treated with several
Fish Physiol Biochem (2013) 39:431–457 441
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doses of b-glucans restricted the growth of Vibrio
algynolyticus, Vibrio splendidus, and Escherichia
coli, but the antibacterial activity modulation was
limited to clams (Costa et al. 2008).
Yeast and yeast subcomponents
as immunostimulant
Yeast and yeast components, that is, glucans and
mannans have been reported to boost immunity in
many fish species. Brewers yeast (Saccharomyces
cerevisiae) is a natural product from the brewing
industry and contains various compounds such as
glucans, nucleic acids as well as mannan oligosac-
charides, capable of enhancing immune responses
(Ortuno et al. 2002) and growth (Lara-Flores et al.
2002) of various fish species. Experimental supple-
mentation of yeast and yeast subcomponents (YYS)
was used to study the physiological performance of
juvenile channel catfish, Ictalurus punctatus. Exper-
imental diets for 4 weeks followed by 2 weeks on
control diet showed no response on the growth
performance, hematology, or immune function but
partial resistance to stress was observed on exposure to
low-water stress with no improved resistance against
E. ictaluri (Welker et al. 2007). Similarly, Nile tilapia
showed minimal effect on immune function and no
effect on growth and mortality when challenged with
Streptoccocus iniae (Shelby et al. 2007). YYS fed Nile
tilapia juveniles did not show any effect on growth,
serum component, antibody response, and resistance
to Streptoccocus iniae and Edwardsiella tarda (Shelby
et al. 2009). Oral administration of yeast cell wall
(YCW) from Saccharomyces cerevisiae in Labeo
rohita against bacterial pathogen, Aeromonas hydro-
phila, was studied by feeding them on experimental
diet for 15 days and then switched back to control diet.
Challenging them with intraperitoneal injection of
virulent A. hydrophila showed low mortality and
enhanced immunostimulatory and protective effect
(Pal et al. 2007), whereas feeding YCW showed
minimum protection against columnaris disease
caused by Flavobacterium columnare in early life
stages of Labeo rohita (Kunttu et al. 2009). Li and
Gatlin (2003) established the beneficial effects of
partially autolyzed brewers yeast on immune
responses of hybrid striped bass and resistance to
S. iniae infection.
In vitro studies with spent brewer’s yeast b-glucan
(BYG) showed enhanced phenoloxidase activity, the
same was demonstrated on oral administration of BYG
in diet for 3 days in black tiger shrimp (Suphantharika
et al. 2003) and improved hemocytes number and the
bacterial killing activity against the pathogen Vibrio
(Thanardkit et al. 2002). Pacific White shrimp
fed with diet containing inactive yeast cell wall
showed no significant difference in weight, survival,
and growth rate, but showed better effects on immune
parameters (total and granular hemocyte count, bac-
terial clearance) compared to control (Chotikachinda
et al. 2008).
b-Glucan with other immunostimulants
Combinations of b-glucan with other immunostimu-
lants are equally effective in improving immunity
against antigens. b-glucan plus O-antigen-treated
turbot leukocytes destroyed an avirulent strain of
V. damsela efficiently and increased respiratory burst in
leukocytes on 7 days post-infection. Lysozyme activ-
ity in serum of fish injected with glucan and O-antigen,
alone or in combinations, was enhanced compared to
the control (Santare
m et al. 1997). Administration of
b-glucan and O-antigen of Vibrio damsela enhanced
several non-specific immune responses in turbot
(Scophthalmus maximus). Immune response of seven
different immunostimulants in Coho Salmon to Aer-
omonas salmonicida was studied by injecting them
along with bacterin. Fish fed with lentinan, formalin-
killed Renibacterium salmoninarum cells, and Vita-
Stim-Taito for 27 days and challenged with Aeromo-
nas salmonicida by cohabitation and immersion
method showed continued increase in the protection
(Nikl et al. 1991). In a study, combined effect of
b-glucan, ascorbic acid (Aa), and a-tocopherol (a-T) was
performed on sea bass. Initially, fishes were adopted to
diet supplemented with Aa, a-T for 5 weeks but later
moved to diet containing b-1, 3/1, 6-glucan and high
dose of Aa and a-T for 2 weeks in every 3 months.
Immune parameters such as alternative pathway of
complement activation and lysozyme activity were
enhanced in experimental fish (Bagni et al. 2008).
Vaccinated juveniles of red tail black sharks
(Epalzeorhynchos bicolor) were fed with b-glucan or
nucleotide for 24 days and then infected with S. iniae
by intracoelemic injection. Both vaccinated and
442 Fish Physiol Biochem (2013) 39:431–457
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non-vaccinated fishes were fed with glucan or nucle-
otide showed low mortality compared to control but no
difference was noticed among fishes fed with b-glucan
and nucleotide (Russo et al. 2006). b-1,3/1,6-glucan
from chrysolaminaran was fed to two sets of Atlantic
cod (Gadus morhua L.) larvae. In one experiment,
effect of microalgal glucan on immunity was com-
pared with MacroGard (Yeast glucan) and the results
showed increased survival rate in fish fed with
microalgal glucan compared to control, but Macro-
Gard had no significant effect on survival. In another
experiment, fish fed with microalgal glucan showed
positive growth rate during weaning to dry feed
compared to high alginate (Mannuronic acid) fed fish
(Skjermo et al. 2006).
b-Glucan had shown its positive effects on growth,
survival, and immune response of fish species against
infectious pathogens alone or in combination with
other immunostimulants or probiotics. Effect of b-1,3/
1,6 glucan and probiotics (Vibrio alginolyticus)
inclusion in shrimp (Penaeus vannamei) larva at three
different stages early, middle, and late was studied.
Shrimp larva fed with probiotics and b-1,3/1,6 glucan
in larviculture, where b-1,3/1,6 glucan showed highest
survival against WSSV in early stage compared to
other stages. Probiotics showed negative effect on
plasmatic protein (PP), but increased antibacterial
activity (AA) and THC. However, b-1,3/1,6 glucan
showed negative effect on the O
generation. In
challenge study, interaction between b-1,3/1,6 glucan
and probiotics was found beneficial for improving
immune parameters (Rodregeuz et al. 2007). Simi-
larly, intramuscular injection of inactivated WSSV
alone or in combination with b-1, 3-glucan and
inactivated Vibrio penaeicida was given to Kuruma
shrimp (P. japonicus). Shrimps were challenged by
intramuscular injection of WSSV on the 10th and 30th
day of post-vaccination with formalin-inactivated
WSSV where no significant difference in mortality
was observed when shrimp was treated with inacti-
vated WSSV alone. However, survival was signifi-
cantly improved in treatment group containing
inactivated WSSV together with b-1, 3-glucan or
inactivated Vibrio penaeicida (Namikoshi et al. 2004).
This result corroborates the finding that glucan and
inactivated Vibrio enhance the resistance against
Vibrio as well as WSSV infections (Sung et al.
1994; Teunissen et al. 1998; Sritunyalucksana et al.
1999). Formalin-killed V. harveyi (bacterin) and
Carboxymethyl b-glucan (CMBG) was administrated
to black tiger shrimp, Penaeus monodon to protect
against V. harveyi infection (Klannukarn et al. 2004).
In one case, bacterin and CMBG were provided with
feed to fishes for 10 days, and in other case, their
combination was used in feed for 2 months in
commercial ponds. The relative percent survival
(RPS) and shrimp hemolymph parameters studies
were demonstrated that bacterin and CMBG can
induce internal defense and provide protection against
V. harveyi when given separately rather than in
combinations. They also determined the mechanism
of cellular and humoral protections in shrimp.
Feeding b-glucan- or vitamin C-containing diet to
Litopenaeus vannamei juvenile enhanced the growth
and increased numbers of blood cells after salinity
shock in fish fed with b-glucan and decreased in Vit C
fed shrimp. Decrease in the proPo activity was
observed in both b-glucan and Vit C fed shrimps,
but the proPO granular cell ratio was enhanced in Vit
C and decreased in b-glucan fed shrimp (Lopez et al.
2003). Immersion of juvenile of American white
shrimp (Litopenaeus vannamei) for 6 h out of 1, 3, 6 h
in aerated sea water along with b-glucan and sulphated
polysaccharide solutions showed increased superox-
ide anion (SOD) activity in hemocytes and muscle
compared to control or untreated samples. The total
hemocyte counts (THC) were decreased in first 24 h of
post-challenge with immunostimulants, but THC and
total soluble hemocyte protein reached to its normal
value after 48–120 h, thus showing induced immuno-
stimulatory activity in muscle and hemocytes (Campa-
rdova et al. 2002).
Dosage of b-glucan in aquaculture
Dosages of immunostimulant given to aquatic organ-
isms play an important role in the stimulation of
immune response. It is essential to know correct
dosages of immunostimulants and appropriate admin-
istration route to be followed to achieve the desired
results. Steam pellet diet containing 0, 0.01, 0.1, and
1.0 % VST (VitaStim-Taito) was fed for 7 days, and
bath challenged with Aeromonas salmonicida (As)
vaccine. Diet containing 0.1 and 1.0 % VST resulted
in significant protection against the Aeromonas
salmonicida when challenged through oral adminis-
tration. In the immersion trials, vaccine and VST were
Fish Physiol Biochem (2013) 39:431–457 443
Author's personal copy
administered either alone or in combinations. Follow-
ing bath challenge with virulent at 21 day of post-
vaccination, no significant protection was noted in any
of the groups tested, indicating that VST was inactive
as an immunopotentiator by this route (Nikl et al.
1993). A diet containing 0.5 g b-1.3/1.6-glucan
(Macrogard)/100 g of pellets was fed to rainbow trout
(Oncorhynchus mykiss) daily for a week and were
immunized by immersing them in anti-Yersinia ruck-
eri vaccine. This resulted in an increased number of
antibody-secreting cells (ASC) and specific Ig levels
in serum, thus enhanced the effectiveness of Yersinia
ruckeri vaccine in fish (Siwicki et al. 2004). However,
feeding them with 0.1 % b-glucan for 4 weeks and
exposing to 2 h of transportation stress showed
elevated innate immune response (phagocytosis and
oxidative radical production) in treated fish and helped
to prevent negative effect of stress and protection
against Flexibacter columnaris. Stress-induced ele-
vated cortisol levels in plasma and hyperglycemia
were lowest in 0.1 % fed b-glucan group (Jeney et al.
1997). Multiple injections of barley b-glucans at
different dosages 0, 5, 10, 15 mg/kg of body weight
were used to enhance the immune response and
disease resistance against Aeromonas hydrophila and
Edwardsiella tarda in Labeo rohita fingerlings. The
results showed that injection of glucan 10 mg/kg body
weight, three times, was advocated to enhance the
immune response (Misra et al. 2006a). Likewise
b-glucan at a dose of 250 mg/kg diet was recom-
mended for enhancing immunity, growth, and survival
against opportunistic pathogens such as Aeromonas
hydrophila and Edwardsiella tarda in rohu. Immune
parameters (leukocyte count, phagocytic ratio, phag-
ocytic index, lysozyme activity, complement activity
and serum bactericidal activity) rose to its highest
levels on 42 days (Misra et al. 2006b). Oral admin-
istration of three barley genotypes with different
quantity of b-glucan low (38 g/kg), average (52 g/kg),
high (82 g/kg) showed decrease in the growth but
increase in the IHNV neutralizing antibody and
enhanced survival against IHNV; however, respiratory
burst, lysozyme, TNF-a showed no response. Survival
rates in fish fed with average or high dose of barley
glucan were similar to yeast glucan (MacroGard) at
2 g/kg fed fish (Sealey et al. 2008).
Efficacies of different routes (intraperitoneal injec-
tion, bathing, and oral administration) were tested on
Cyprinus carpio, where fish was fed with b-glucan and
LPS to study survival and immune response on
challenging with Aeromonas hydrophila. Intraperito-
neal injection showed 100 % relative percentage
survival (RPS) at all concentrations of b-glucan,
whereas oral administration showed high RPS at
higher concentrations (1 % b-glucan ? 0.25 % LPS),
but bathing did not improved RPS levels. Minimum
dose (100 mg b
-glucan ? 10 mg LPS) per fish had a
significant increase in total blood leukocyte, neutro-
phils, monocytes counts, and superoxide anion activ-
ity. Neither dose nor the route of administration of
compounds had a significant effect on increase in
expression of interleukin-1 beta mRNA and on
classical and alternative complement pathways (Selv-
araj et al. 2006). Baker’s yeast b-glucan was admin-
istered though same routes in different concentrations
100, 500, 1,000 lg, but intraperitoneal injection of
500 lg showed enhanced RPS, whereas bathing and
oral administration showed no effect on RPS. All the
three concentrations showed increase in total blood
leukocyte counts, neutrophils, monocytes, and ele-
vated expression of interleukin-1beta mRNA on
7th day in fish injected with glucan (Selvaraj et al.
2005). Immunized and non-immunized Nile tilapia
were fed with different concentrations of b-glucan 0,
50, 100, 200 mg for 14 weeks. b-glucan, immuniza-
tion, and their combination showed no effect on
growth and survival after 10 weeks, but fish fed with
100 and 200 mg b-glucan showed partial decrease in
feed efficiency ratio (FER) compared to 50 mg glucan
fed fish. Immunized fish showed reduced percentage
mortality (PM) and increased RPS against S. iniae;
moreover, their combination had no effect on PM and
RPS (Whittington et al. 2005).
Large yellow croaker diets supplemented with 0 %
(control), 0.09 % (low), and 0.18 % (high) of b-1, 3
glucan were fed for 8 weeks, low glucan levels
enhanced fish growth, while high levels significantly
enhanced the lysozyme activity. Respiratory burst
activity in head kidney macrophage was enhanced
with low concentration of b-glucan. Overall growth,
lysozyme, phagocytosis, respiratory burst, and pro-
tection against V. harveyi were enhanced but there was
no effect on alternative complement pathway (Ai et al.
2007). Feeding 0 and 10 g whole wild yeast or fks-1
mutant strain per kg diet for 2, 4, and 6 weeks to
gilthead seabream (Sparus aurata L.) showed
decrease in serum peroxidase and complement activity
after 6 weeks and increase in lysozyme activity after
444 Fish Physiol Biochem (2013) 39:431–457
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2 weeks. Phagocytosis was increased to a significant
degree during all the time, but only with fks-1 strain-
supplemented diet, while respiratory burst activity and
natural cytotoxicity (after 4 and 6 weeks) were
enhanced with either yeast strain; moreover, intracel-
lular content was not changed with either of the strain
guez et al. 2003). Head kidney phagocytic cells
from blue gourami (Trichogaster trichopterus)
injected intraperitoneally with laminaran b-1,3-glucan
in different dosages of 5, 10, 20, and 40 mg/kg were
studied for chemiluminescent response (CL). Fish
injected with 20 mg/kg dose showed enhanced CL and
resistance to A. hydrophila up to 22 days. However, 5,
10, and 40 mg/kg dose showed enhanced CL for
shorter duration up to 14 days and low resistance to
Aeromonas hydrophila. Fish treated with 10 mg/kg
glucan plus formalin-killed bacteria showed higher
CL response and resistance compared to bacteria
injected fish. Fish fed with diet containing 20 mg/kg of
laminaran plus bacteria showed higher response and
resistance at 22 days. Thus, 20 mg/kg of laminaran
can protect the fish effectively from A. hydrophila up
to 29 days (Samuel et al. 1996). Laboratory developed
feed stimulant, BAISM, and dietary supplementation
of b-1,3-glucan were used to study their effect as feed
additives on juvenile olive flounder, Paralichthys
olivaceus (Yoo et al. 2007), and it was concluded from
the study that optimum level of b-1,3-glucan and
BAISM could be approximately 0.1 % b-1,3-glu-
can ? 0.9 % BAISM (G0.1B0.9) of diet for better
growth and survival.
Similarly, feeding 0, 0.5, 1.0 % yeast culture
supplement diamond VXP YC to pacific white shrimp
(Litopenaeus vannamei) daily for 4 weeks has shown
protection against bacterial diseases (Burgents et al.
2004). Oral administration of fungal polysaccharide
schizophyllan at a dose of 50 or 100 mg/kg body
weight per day showed higher survival rates after
challenging with the virus (RV-PJ) compared to the
control. Phagocytic activities were found to be higher
in schizophyllan fed shrimps. These results indicate
that schizophyllan can confer effective protection
against viral infection by increasing antiviral immune
responses in invertebrates (Itoh 1997). Similarly,
Schizophyllum commune glucan fed for 40 days to
grass prawn in indoor and outdoor rearing tanks.
Irrespective of rearing tanks, enhanced survival com-
pared to control was recorded in shrimps fed with
b-glucan at a dose of 20 g/kg diet. Hemocyte phagocytic
activity, cell adhesion, and superoxide anion produc-
tion were also elevated (Chang et al. 2000). The same
glucan (0, 1, 2, 10, 20 g/kg diet) was fed to Penaeus
monodon for 20 days and challenged with WSSV
injection. Shrimps fed with 10 g/kg glucan showed
highest survival (P \ 0.05) than other groups,
whereas hemolymph THC, PO, O
, and SOD were
dropped initially in all group except in 10 g/kg fed
glucan group, on challenging and reached normal later
(Chang et al. 2003). Different concentrations of
marine yeast b-glucan (0.05, 0.1, 0.2, 0.3, 0.4 g/100
gm) were fed to post-larvae of Indian white shrimp for
21 days. Larval survival was observed against oral
administration of white spot syndrome virus and
shrimps fed with 0.2 % glucan showed maximum
survival, while in second experiment, 0.2 % glucan
was fed at different days intervals and study showed
that 0.2 % glucan when given once every 7 days
recorded best result (Sajeevan et al. 2009). Post-larvae
of tiger shrimp were immersed in aerated b-glucan
suspension for 3 h in four different concentrations
0.25, 0.5, 1.0, 2.0 mg/ml. Enhanced growth of larvae
was observed in 0.5, 1.0, and 2.0 mg/ml b-glucan
suspension, but no response in 0.25 mg/ml glucan was
observed. After 43 days when larvae were immersed
in bacterial suspension of Vibrio vulnificus cells
(5 9 10
CFU/ml for 12 h), increased protection
against the pathogen along with enhanced phenoloxi-
dase activity in shrimp hemocyte was reported (Sung
et al. 1994). Effective doses of various b-glucan used
in aquaculture have been summarized in Table 2.
b-Glucan and immunocompromised fish
Several experiments have been carried out to study the
effect of b-glucan on immunocompromised fish in
enhancing resistance to infectious pathogens. Immu-
nity of immunocompromised fish is reduced using
certain toxins or chemicals. Effect of aflatoxin B
b-1,3-glucan, and their combination on non-specific
immunity and disease resistance was studied (El-
Boshy et al. 2008). Dietary supplementation of
aflatoxin (AFB
) in fish showed reduced immunity
with affected biochemical parameters related to organ
damage. Feeding 0.5 % b-1,3-glucan to immunocom-
promised (200 lg/feed aflatoxin B1) Nile tilapia for
21 days showed enhanced resistance against S. iniae
and improved non-specific immunity levels compared
Fish Physiol Biochem (2013) 39:431–457 445
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Table 2 Effective doses of various b-glucan used in aquaculture
Types of b-glucan Dosages used Route of
Species studied Remarks References
1. b-Glucan
5, 2 and 0.5 mg/ml Intraperitonial
Danio rerio 5 mg/ml showed reduction in the mortality to A.
hydrophilla and increased myelomonocytic cell
population. Modulation of IFN-c and chemokine
expression in kidney
guez et al.
2. Marine yeast glucan 0.2 g/kg feed once in a
Oral Fenneropenaeus
Improved survival against WSSV Sajeevan et al.
3. Brewer’s yeast b-glucan 0.2 % (w/w) for
3 days
Oral Penaeus monodon Enhanced phenoloxidase activity Suphantharika
et al. (2003)
4. Brewer’s yeast b-glucan 0.2 % (w/w) for
3 days
Oral Penaeus monodon Increased number of haemocytes and the bacterial
killing activity against Vibrio harveyi
Thanardkit et al.
5. Inactive yeast cell wall 1 and 2 g/kg feed Oral Litopenaeus vannamei Improved immune parameters Chotikachinda
et al. (2008)
6. Inactive yeast cell wall 1 and 2 g/kg feed Oral Litopenaeus vannamei No effect on weight, survival and growth rate Chotikachinda
et al. (2008)
7. b-glucan 0.1 % of feed Oral Oncorhynchus mykiss Lowest cortisol levels in plasma against
transportation stress
Jeney et al. (1997)
8. Barley b-glucans 5, 10, 15 mg/kg of
body weight
Injection Labeo rohita 10 mg/kg body weight, three times showed
enhanced immune response and disease resistance
against Aeromonas hydrophila and Edwardsiella
Misra et al.
9. b-glucan 250 mg/kg feed for
42 days
Oral Labeo rohita Enhance immunity, growth and survival against
opportunistic pathogens such as Aeromonas
hydrophila and Edwardsiella tarda
Misra et al.
10. b-glucan 38 g, 52 g and 82 g/kg
Oral Oncorhynchus mykiss Showed decrease in the growth but enhanced
survival against IHNV
Sealey et al.
11. Baker’s yeast b-glucan 100, 500, 1,000 lg/
Injection Cyprinus carpio 500 lg showed enhanced RPS and all three showed
increase in total blood leukocyte counts,
neutrophils, monocytes and elevated expression of
interleukin-1beta mRNA
Selvaraj et al.
12. b-1, 3 glucan 0.09 and 0.18 % Oral Pseudosciaena crocea Low glucan levels enhanced fish growth, while high
levels significantly enhanced the lysozyme activity
Ai et al. (2007)
13. Wild yeast or fks-1
mutant strain
10 g/kg diet for 2, 4
and 6 weeks
Oral Sparus aurata L. Decreased serum peroxidase and complement
activity after 6 weeks and increased lysozyme
activity after 2 weeks
guez et al.
14. VXP YC 0.5 and 1.0 % Oral Litopenaeus vannamei Protection against bacterial diseases Burgents et al.
446 Fish Physiol Biochem (2013) 39:431–457
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Table 2 continued
Types of b-glucan Dosages used Route of
Species studied Remarks References
15. Fungal polysaccharide
50 or 100 mg/kg body
Oral Litopenaeus vannamei Higher survival rates after challenging with the virus
Itoh (1997)
16. Schizophyllum
commune glucan
20 g/kg diet Oral Penaeus monodon Enhanced survival and immunity Chang et al.
17. Schizophyllum
commune glucan
1, 2, 10, 20 g/kg diet Oral Penaeus monodon 10 g/kg glucan showed highest survival and
immunity against WSSV infection
Chang et al.
18. b-glucan 0.25, 0.5, 1.0, 2.0 mg/
Immersion Penaeus monodon
Enhanced growth and protection to Vibrio vulnificus
was observed in 0.5, 1.0 and 2.0 mg/ml glucan
Sung et al. (1994)
Dosages of b-glucan with other immunostimulants
19. b-glucan and nucleotide 1 g/kg ?2 g/kg feed Oral Epalzeorhynchos
bicolor juveniles
Increased resistance against S. iniae Russo et al.
20. Coomercially available
b-glucan, VitaStim-
Taito (VST)
0.01, 0.1, and 1.0 %
for 7 days
Oral and
0.1 and 1.0 % VST resulted in significant protection
against the Aeromonas salmonicida
Nikl et al. (1993)
21. b-1.3/1.6-glucan
(Macrogard) and
immunized by anti-
yersinia ruckeri
0.5 g/100 g of pellets
for a week
Oral Oncorhynchus mykiss Increased number of antibody secreting cells (ASC)
and specific Ig levels in serum
Siwicki et al.
22. b-glucan and LPS 1 % ? 0.25 % Oral Cyprinus carpio Improved RPS to Aeromonas hydrophila Selvaraj et al.
23. b-glucan and LPS 100 mg ?10 mg/fish Injection Cyprinus carpio Increased total blood leukocyte, neutrophils,
monocytes counts and superoxide anion activity
Selvaraj et al.
24. b-glucan and
50, 100, 200 mg/kg
Oral Oreochromis niloticus No effect on growth and survival after 10 weeks Whittington et al.
25. Head kidney phagocytic
cells and Laminaran
5, 10, 20 and 40 mg/
kg feed
Injection ? oral Trichogaster
20 mg/kg feed showed enhanced chemiluminescent
Samuel et al.
26. BAISM and b-1,3-
0.9 % ? 0.1 % Oral Paralichthys olivaceus Better growth and survival Yoo et al. (2007)
Fish Physiol Biochem (2013) 39:431–457 447
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to AFB
non-treated fish. An artificial immunosup-
pressor, cyclophosphamide was used in Asian catfish,
Clarias batrachus to make them immunocompro-
mised, the cyclophosphamide fed fish showed lower
respiratory burst, myeloperoxidase, phagocytic and
hemagglutination activity in blood; however, use
of b-1,3-glucan as feed supplement resulted in
enhanced immune response in immunocompromised
fish. Superoxide anion, myeloperoxidase, phagocytic
activity, and hemagglutination were also increased
(Kumari and Sahoo 2006b). In same immunocompro-
mised fish, use of b-glucan enhanced antibody
production and protection against Aeromonas hydro-
phila when challenged with formalin-killed A. hydro-
phila bactericin (Kumari and Sahoo 2006a). Feeding
b-1,3 glucan to immunocompromised Labeo rohita
for 7 days showed significant enhancement in the
resistance against A. hydrophila. The non-specific
immunity levels such as leukocyte number, phago-
cytic index, and serum bactericidal activity were also
enhanced compared to non-treated AFB
-exposed fish
(Sahoo and Mukherjee 2001). Aflatoxin B
immunocompromised Indian Major Carp, vaccinated
with formalin-killed Edwardsiella tarda, showed
enhanced specific immunity and reduced mortality,
but failed to enhance specific immunity and protection
in healthy fish. The increased bacterial agglutination
titer and reduced mortality by feeding b-1,3-glucan in
healthy vaccinated fish compared to control indicate
glucan as an potent immunostimulant (Sahoo and
Mukherjee 2002).
b-Glucan as adjuvant
Adjuvants are compounds that enhance the specific
immune response against co-inoculated antigens. Oils
and bacterial lipopolysaccharides have been shown to
induce elevated antibody production when adminis-
tered in fish, but oil emulsions as adjuvant may lead to
side effects such as adhesions of internal organs,
reduced growth, skeleton deformations, etc. (Berg
et al. 2006). Effect of b-1,3-glucan on enhancing the
specific immune response when added together with
immunogens and administered through any route
(injection, bath or dietary incorporation) in fish was
demonstrated by Anderson (1997). Yeast b-glucan
(YBG) has been used as adjuvant from past many
years in aquaculture; Vibrio damsela vaccine was
injected prior, together, and post-application of yeast
(Saccharomyces cerevisae) b-1,3-glucan in turbot
(Scophthalmus maximus L.). The highest activity
among all the immune parameters was obtained when
glucans were injected after the bactericin application.
Finding of this study indicates that the sequence of
glucan administration is critical when used as a
vaccine adjuvant (Figueras et al. 1998). b-glucan of
same species showed adjuvant effect on antibody pro-
duction, when pretreated by injecting 100–1,000 lg
glucan/fish, resulted in enhancing the production of
highest antibody titer against A. hydrophila following
vaccination in Cyprinus carpio (Selvaraj et al. 2005).
YBG along with A. salmonicida vaccine showed
protective response in Atlantic salmon (Rørstad 1993);
moreover, similar protective effect was also observed
against furunculosis disease (Midtlyng et al. 1996).
Feeding Japanese flounder with formalin-killed bac-
tericin E. tarda along with curdlan, a b-glucan showed
no antibody response, but the survival rate was higher
compared to the control fish when challenged with
E. tarda (Ashida et al. 1999). Mushroom glucan as
adjuvant in conjunction with a formalin-killed Aero-
monas hydrophila vaccine in Catla catla showed
significant enhancement in antigen-specific prolifera-
tion, ‘macrophage activating factor’ (MAF), and
antibody production leads to increased protection
against A. hydrophila (Kamilya et al. 2006). b
glucan adjuvant in the Aeromonas vaccine enhanced
production of antibodies to whole or outer membrane
of bacteria and LPS and stimulated production of
protein reactive antibodies, but no sufficient protection
and survival against challenge with Aeromonas sal-
monicida was observed (Aakre et al. 1994). Injection
of 100 mg b-glucan and 10 mg LPS/fish had an
adjuvant effect on antibody production, resulted in
higher antibody titer against A. hydrophila following
vaccination, and enhanced resistance in carp (Selvaraj
et al. 2006).
In contrast, some reports discuss on negative effect
of b-glucan adjuvant. Oral administration of yeast
b-glucan following Streptococcus iniae vaccine
showed no protection against S. iniae in Oreochromis
niloticus (Whittington et al. 2005) and against Strep-
tococcus bacterin in turbot (Romalde et al. 1999).
When yeast b-glucan was co-injected along with
Pasteurella piscicida vaccine, the glucan showed a
negative adjuvant effect in yellow tail (Kawakami
et al. 1998).
448 Fish Physiol Biochem (2013) 39:431–457
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Glucans as prebiotics
Prebiotics are non-digestible feed ingredients that
benefit the host by stimulating growth and activity of
beneficial bacteria in gastrointestinal (GI) tract (Gib-
son and Roberfroid 1995). The main advantage of
prebiotics over probiotics is that they are natural feed
ingredients thus regulatory control over dietary sup-
plementation is limited (Gatelin III et al. 2006). Use of
most of the antibiotics is banned due to potential
development of antibiotic-resistant bacteria (FAO
2002), presence of their residues in seafood, destruc-
tion of microbial populations in the aquaculture
environment, and suppression of the aquatic animal’s
immune system. Report has shown that antibiotics
may impede growth and feed efficiency by killing
intestinal microflora and thus reducing amino acid
utilization by the host in some animal species (Rawles
et al. 1997). These adverse effects and cost of
antibiotics to overcome the diseases encouraged
investigators to explore alternatives and application
of prebiotics is one among such options.
, a commercial prebiotics, is a mixture
of partially autolyzed brewer’s yeast, dairy components,
and dried fermentation products. It has shown enhanced
growth performance and disease resistance in hybrid
striped bass (Gatlin and Li 2004;LiandGatlin2004,
2005). Feeding GroBiotica-A
-supplemented diet
enhanced protein and organic digestibility in striped
bass (Gatlin et al. 2006) and in rainbow trout improved
resistance against different pathogens (Sealey et al.
2007). Dairy yeast prebiotics in golden shiners (Notem-
igonus crysoleucas) have enhanced the growth and
survival (Lochmann et al. 2009), while mortality against
Flavobacterium columnare was reduced significantly
(Sink and Lochmann 2008). The survival, growth, and
immune response were enhanced in juvenile western
king prawns (Penaeus latisulcatus) by feeding Bio-
MosÒ in combination with b-1,3-
D-glucan (Hai and
Fotedar 2009). However, it improved the survival,
health status, and immunity of marron, Cherax tenuim-
anus, against bacterial infection and under stress
conditions with NH
exposures (Sang et al. 2009).
Use of Fermacto prebiotic improved growth and
feed efficiency ratio of common carp (Cyprinus
Carpio) fry (Mazurkiewicz et al. 2008). Feeding
crucian carp (Carassius auratus) with prebiotic
xylooligosaccharides showed no response on the
growth and survival, but difference in the protease
and amylase activity compared to the control was
reported (Xu et al. 2009). Feeding short-chain fruc-
tooligosaccharide (scFO) supported growth of micro-
bial flora of GI tract and enhanced the immunity in
Pacific white shrimp (Li et al. 2007). Similarly
fructooligosaccharide and mannan oligosaccharides
showed positive effect on growth of Atlantic salmon
(Salmo salar) (Grisdale-Helland et al. 2008), and
mannan oligosaccharide showed enhanced salinity
tolerance and development of micovilli in gut of cobia,
Rachycentron canadum larva (Salze et al. 2008).
Surging human population has created spiraling
demand of food, which needs to be met through
innovative approaches without ignoring the context of
human health and environmental sustainability. In the
background of such concerns, b-glucan has tremendous
potentials to be used in fish culture, disease manage-
ment, and fish products development through biotech-
nical approach. An agent for prophylaxis and disease
management, b-glucan, imparts immunity against var-
ious fish pathogens. Research reports have shown
b-glucan dosages, quality, route, and time of adminis-
tration and duration of treatments play significant role
in enhancing various parameters related to growth,
survival, and immunity. There is a need to give
emphasis on optimizing the dosage and improve the
quality of b-glucan to be used in different fish species.
The adjuvant effect of b-glucan needs to be further
addressed. The possible use of b-glucan from other
sources like seaweed, plants, etc., should also be
explored. Application of b-glucan as prebiotics in
aquaculture is another emerging avenue, which requires
further attention. So, further research on unexplored
roles of b-glucan should be undertaken to adopt
b-glucan as a potent immunostimulant in aquaculture.
Acknowledgments The authors are grateful to the Director,
Central Institute of fisheries Education, Versova, Mumbai-61
(India) for his constant support, encouragement, and providing
facilities for the study.
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