Fish & Shellfish Immunology (2008) 25, 384e396

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/fsi

REVIEW

-glucans as conductors of immune symphonies

Roy A. Dalmo*, Jarl Bgwald **
Department of Marine Biotechnology, University of Troms, N-9037 Troms, Norway
Received 17 December 2007; revised 17 April 2008; accepted 18 April 2008
Available online 25 April 2008

KEYWORDS
Immunostimulants;
-glucan;
Adaptive immunity;
Th1;
Th2;
Th17;
Adjuvants;
Vaccines;
Dectin-1;
Toll Like
receptors;
Shrimp

Abstract The use of immunostimulants has received increased attention due to the discovery
of Toll-like receptors (TLR) or/and pattern recognition receptors (PRR). These receptors have
been found to bind molecules from a range of pathogens including self-molecules. When cell
damage has occurred many of the released molecular structures act as so-called danger
signals possessing pathogen-associated molecular patterns (PAMP). These danger signals often
consist of repeating molecular moieties yielding high molecular weight compounds. Examples
are -glucans and CpG containing DNA, but some danger signals possess low molecular weight
structures. It has been found that the PRR bind unit structures of PAMP, and that PAMP-binding
involves several other humoral and cell membrane proteins, exemplified by the more or less
simultaneous LPS recognition displayed by MD-2, CD-14 and TLR4 on the cell membrane. Also,
the binding of -glucans has been shown to include several different cell membrane receptors.
Several immunostimulants are commercially exploited in aquaculture as feed additives. This
applies to -glucans, alginates and nucleotides. Despite their use as feed additives no targeted
approach has been conducted to include PAMP as adjuvants in fish vaccines. Interestingly, most
of the PAMP studied activate antigen-presenting cells together with nave T cells into dendritic
cells and Th1 or Th2 cells [1]. In turn, this may activate Th1 and Th2 immune responses with production of Th1 or Th2 signature molecules such as IFN-gamma and IL-4, respectively [2e4]. This
review will mainly focus on binding characteristics of -glucans, their effects on T helper cell differentiation, effects on functional levels, gene expression profiles and application of the commonly used -glucan in the aquaculture sector. In addition, -glucans show promises in shrimp
aquaculture by inducing disease resistance, this review will also highlight the use and the effects
of -glucans in experimental models.
2008 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: 47 77 644482; fax: 47 77 645110.

** Corresponding author. Tel.: 47 77 646069; fax: 47 7764 5110.
E-mail addresses: roy.dalmo@nfh.uit.no (R.A. Dalmo), jarlb@nfh.uit.no (J. Bgwald).
1050-4648/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fsi.2008.04.008

-glucans in aquaculture

Introduction
A number of immunostimulants has molecular architecture
possessing repeating units of a certain moiety such as
glucose in -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, PAMP, if they
initiate inflammatory responses. Interestingly, most of the
PAMP studied activate antigen-presenting cells together
with naive T cells into dendritic cells and T helper cells [1e
4]. During microbial breakdown/degradation, a number of
PAMP may be released that would initiate inflammatory responses upon receptor binding and intracellular activation
of signal transducers and transcription factors. The exact
composition of released danger signals would be decisive
for the out-put gene transcription leading to synthesis and
release of pro-inflammatory cytokines. Immunostimulants
have been used as feed additives for many years in aquaculture, and yeast -glucan may be the one with the longest
track record. Homo-polysaccharides, consisting of one sugar,
are described by use of the suffix an. Thus a poly-glucose
(poly-glucopyranose) is termed glucan. Other carbohydrates may be hetero-polysaccharides that may contain
several different constituent residues. (1,3)-D-glucans
(-glucans) are the most commonly used term for homopolysaccharides that has (1,3)-D-linkages in the backbone,
and may also possess -D-glucosidic linkages at position 6 in
different, often repeating units (branches). In nature, -glucans are widespread and are found in plants, algae,
bacteria, yeast and mushrooms. -glucans possess differences in molecular weights and degree of branching. Yeast
-glucan is a particulate carbohydrate that consists of glucose and mannose and is a major constituent of the cell
membrane. Of the major fish feed producers, Skretting
and Ewos include yeast -glucan in their fish feed assortments, while BioMar includes bioactive alginate.

Growth performance
Besides the information presented in comprehensive review
articles on immunostimulants [5], several new reports have
described the effects of -glucans on growth performance
in fish. It has been found that -glucans such as yeast glucans obtained from Saccharomyces cerevisiae incorporated
in fish feed increase the growth rate of certain species of
fish at certain environmental conditions [6e9]. However,
in other scientific experiments no significant growth
increase has been found after feeding fish with different
-glucans [10e12]. It appears that a growth increase is
dependent on the amount of -glucan incorporated in the
diet, duration of feeding, environmental temperature and
the species under study. To obtain increased growth performance, feeding strategies should be developed for each fish
species with respect to -glucan dose and duration. In addition, soluble or particulate -glucans may confer different
responses since the intestine only absorb soluble -glucans.
There are no indications that particulate -glucans are
taken up by the fish intestine or digested by -glucan
degrading enzymes thereby being nutritious. Feed additives

385
such as bioactive alginates, enriched with mannuronic acid
residues, have also been reported both beneficial and
detrimental with respect to weight increase and protein
turnover in fish larvae and juveniles [13e15]. Dietary nucleotides may induce weight gain in fish although it seems that
it is likely that species and dose dependencies occur
[11,16,17]. Following on, the weight of salmon given formulated feed with 0.01% and 0.03% w/w Aeromonas salmonicida LPS for 62 days exhibited statistically higher weight
compared to control fish. At the same time, the weight of
fish that received 0.1% LPS did not differ from controls
[18]. To date, there are no plausible explanations for the
growth enhancing effects displayed by diets containing
immunostimulants. One hypothesis may include a local
intestinal inflammatory response, after administration
that induces resistance against pathogens - that otherwise
would result in decreased weight gain and maybe disease.
Evidently, to assess the growth enhancing effects of immunostimulants more sophisticated analysis must be companioned. This should be highly feasible due to the advanced
genomic tools available for gene expression analysis.

Immune responses after oral administration

Sea bass (Dicentrarchus labrax) were fed a diet supplemented with 2% -1,3/-1,6-glucan over a 2-week period
every 3 months [19]. The plasma complement activity was
elevated in immunostimulated fish compared with controls.
In another trial, alternative pathway of complement activation and lysozyme activity were both significantly enhanced
in day 15 samples obtained from fish fed -glucan (MacroGard) and alginic acid (Ergosan) after the first out of
four 2-week feeding cycles. The immunostimulants did
not confer any immune modulation measured as elevated
lysozyme levels, increased complement activity, and modulated T and B cell numbers in peripheral blood after the
fish were fed for 35 weeks [11]. The dietary effect of b1,3-glucan on innate immune responses of large yellow
croaker (Pseudosciaena crocea) was studied [8]. The diet
was supplemented with 0.09% and 0.18% b-1,3-glucan and
the feeding trial lasted for 8 weeks. Low concentration of
glucan (0.09%) significantly enhanced the respiratory burst
and phagocytic activity in head kidney macrophages while
the high concentration (0.18%) did not. The serum lysozyme
activity in fish fed diets with both glucan concentrations
were significantly higher than the control, and the 0.18%
glucan diet significantly stimulated the lysozyme activity
compared to 0.09%. No significant differences were seen
in alternative complement pathway activity with any of
the -glucan concentrations (see overview in Table 1). A
commercial -glucan (EcoActiva) was administered as
a feed supplement to the snapper, Pagrus auratus [6].
The immunostimulant increased macrophage oxygen
radical production during the wintertime, but not during
the summer time. In contrast to macrophage activity
EcoActiva did not potentiate neither the classical nor the
alternative complement activity. The results of this study
suggested that it may be favourable to include -glucan
in the feed to snapper during the wintertime both to increase resistance against diseases and to increase growth
rates. Immunomodulation by a yeast -1,3/-1,6-glucan in

386
Table 1

R.A. Dalmo, J. Bgwald

In vivo effects of immunostimulant in different fish species

Agent (glucan)

Fish species

Administration

Effects

Reference

EcoActiva

Pink snapper
(Pagrus auratus)

Oral

Macrophage respiratory
burst[
Growth[

[6]

-glucan

Rohu (Labeo
rohita Hamilton)

Oral

Superoxide anion[
Phagocytosis[
Lysozyme[
Haemolytic complement[
Serum bactericidal activity[
Specific growth[

[7]

-1,3-glucan

Large yellow
croaker (Pseudosciaena
crocea)

Oral

Lysozyme[
Fish growth[
Alternative complement
pathway/
Phagocytosis[
Respiratory burst[
Protection against Vibrio
harveyi[

[8]

-glucan (barley)

Rainbow trout
(Oncorhynchus
mykiss)

Oral

GrowthY
Respiratory burst/
Lysozyme/
TNF-a/
IHNV neutralising
antibody[
Survival IHNV[

[9]

-glucan

Nile tilapia
(Oreochromis
niloticus)

Oral

Lysozyme/
Agglutination antibody
titres/

[10]

Yeast -glucan

Sea bass
(Dicentrarchus
labrax)

Oral

Serum complement
activity[
Serum lysozyme[
Gill and liver heat shock
protein[

[11]

Whole-cell/yeast
subcomponents

Channel catfish
(Ictalurus
punctatus)

Oral

Growth/
Plasma lysozyme/
Spontaneous haemolytic
complement/
Bactericidal activity/
Survival Edwardsiella
ictaluri infection/

[12]

-1,3,1,6-glucan

Atlantic cod
(Gadus morhua)

Oral

-1,3,1,6 yeast glucan

Rainbow trout

Oral

[15]

Oxidative burst/
Pinocytosis/
Lysozyme/
Alternative complement
activation/
Con A lymphocyte
stimulation[
Antibody response[

[20]

(continued on next page)

-glucans in aquaculture

387

Table 1 (continued)
Agent (glucan)

Fish species

Administration

Effects

Reference

-1,3-glucan
(Saccharomyces
cerevisiae) LPS

Carp (Cyprinus
carpio)

Intraperitoneal
/oral

Superoxide anion[
Adjuvant effect[
Classical/alternative
complement/
Resistance Aeromonas
hydrophila[

[26]

-1,3-glucan

Rohu

Oral

Resistance to Aeromonas
hydrophila[
Leucocyte numbers[
Phagocytic index[
Serum bactericidal activity[

[22]

-1,3-glucan

Rohu

Oral

Antibody response[
Resistance against
Edwardsiella tarda[

[23]

-1,3 yeast glucan

Asian catfish
(Clarias
batrachus)

Oral

Antibody response[
Protection against
Aeromonas hydrophila[

[24]

-1,3 yeast glucan

Asian catfish

Oral

Superoxide anion[
Myeloperoxidase[
Phagocytic activity[
Haemagglutination
(immunocompromised)[

[25]

-1,3 yeast glucan

Carp

Intraperitoneal,
bath, oral

Blood leucocyte number[

Superoxide anion[
Kidney IL-1 mRNA[
Antibody production[
Classical and alternative
complement/

[21]

Mushroom glucan

Catla (Catla catla)

Intraperitoneal

Adjuvant[
Antibody response[
Resistance against
Aeromonas hydrophila/

[30]

-1,3,1,6 yeast
glucan

Atlantic salmon
(Salmo salar)

Intraperitoneal

Adjuvant[

[33]

-1,3-glucan

Atlantic salmon
Yellowtail
(Seriola
quinqueradiata)

Intraperitoneal

Adjuvant[

[34]
[38]

-1,3-glucan
(laminaran)

Rainbow trout

Intraperitoneal

IL-11[
IL-12[
IL-6[
C3-1[
C3-2[/Y
C3-3Y

[56]

388
rainbow trout was evaluated with macrophage activities,
lysozyme and complement activation. In addition, antibody
responses were analysed after vaccination with formalinkilled Yersinia ruckeri [20]. The non-specific immune
parameters were evaluated at the end of a 2-week feeding
period and 4 weeks later. A significant enhancing effect of
dietary -1,3/-1,6-glucan was found on concanavalin Ainduced proliferation of lymphocytes and the antibody
response after vaccination against Y. ruckeri. But glucan
showed no effect on oxidative burst, pinocytosis, lysozyme
activity and alternative complement pathway activation.
Carp (Cyprinus carpio) were given -glucan by the oral
route before vaccination with Aeromonas hydrophila. The
antibody titre was slightly increased. Orally administered
glucan did not result in changes of the alternative complement pathway [21]. -Glucan was administered to Labeo
rohita fingerlings through different dietary doses for 56
days. Leucocyte counts and both cellular and humoral
immune parameters were evaluated at 2 weeks intervals
[7]. Administration of the highest dose (500 mg -glucan
per kg diet) resulted in a reduced white blood count
compared to fish fed 250 and 100 mg. Maximum superoxide
anion production and phagocytic activity in head kidney
macrophages were found in fish fed 500 mg and 250 mg
-glucan per kg diet (for 42 days), respectively. Haemolytic
complement activity was significantly higher in fish stimulated with -glucan except the 500 mg per kg dose. The
lysozyme and bactericidal activity were highest at a dose
of 250 mg -glucan per kg dry diet fed for 42 days. It is
known that -glucans and LPS may activate the alternative
pathway of complement. Indeed, an intraperitoneal injection of LPS (e.g., 5 mg LPS kg body weight1) has shown
to be exhaustive of the haemolytic activity of complement
in the spotted wolffish (own observation). On the other
hand, whether activation of haemolytic complement activity following oral administration of immunostimulants is
beneficial in terms of disease resistance remains to be
addressed.

Effects on immunocompromised fish

The dietary effect of -1,3-glucan on immune responses in
healthy and aflatoxin-induced immunocompromised rohu
(L. rohita Hamilton) was investigated. A single injection of
aflatoxin reduced the non-specific immunity as measured
by neutrophil phagocytosis, serum bactericidal activity and
specific immunity against Edwardsiella tarda [22,23]. Feeding of glucan to aflatoxin-immunocompromised fish for 7 days
significantly raised non-specific immunity as measured by
serum bactericidal activity, phagocytic activity and antibody
titres (Table 1). Cyclophosphamide, a multifunctional alkylating agent as well as a cytotoxic drug e a well-known immunosuppressant was used to induce an immunocompromised
state in Asian catfish (Clarias batrachus) [24,25]. The cyclophosphamide treated fish showed lowered level of respiratory burst, myeloperoxidase and phagocytic activities in
blood phagocytes, and a decreased haemagglutination activity. -Glucan delivered as feed supplement significantly
enhanced these immune parameters. Taken together, the
use of, e.g., -glucan may have advantages during immunosuppressive states such as during physiological and

R.A. Dalmo, J. Bgwald

environmental stress. The feed manufacturers are also advising to use feed with immunostimulants during such
circumstances.

Immune responses after -glucan injection

Carp (C. carpio) injected intraperitoneally with -glucan
(100, 500 and 1000 mg) had a significant increase in total
leucocyte counts and an increased proportion of neutrophils and monocytes on day 7 [21]. Classical and alternative
complement activation were unaffected by glucan injection. Anterior kidney macrophages from both control and
glucan-injected fish were cultured and assayed for superoxide anion production, bacterial killing of A. hydrophila and
expression of IL-1 mRNA. Superoxide anion production was
significantly increased in macrophages from glucaninjected fish at all doses. In addition, A. hydrophila was
more efficiently killed by macrophages from the glucanstimulated fish (Table 1). Also, expression of IL-1 mRNA
was higher and dose-dependent in glucan-treated fish.
A combination of -glucan and lipopolysaccharide (LPS)
injected intraperitoneally to carp (100 mg -glucan 10 mg
LPS) had a significant increase in total blood leucocyte
counts and an increase in the proportion of neutrophils
and monocytes [26]. Superoxide anion production by
macrophages was also elevated. Classical and alternative
complement pathways were unaffected by the glucan.
Also, expression of IL-1 mRNA was not elevated in the
stimulated fish.

Adaptive immunity
T helper cells and differentiation
During an immune response, professional antigen presenting cells (APC) endocytose, e.g., bacteria or viruses, that
undergo processing, then travel from the site of infection to
other lymphoid tissues/organs where antigen presentation
occurs. This allows nave CD4 T cells that express specific
TcRs against the peptide/MHC complex to be activated.
When a Th cell encounters and recognises the antigen on
an APC, the TcReCD3 complex binds strongly to the peptideeMHC complex present on the surface of professional
APC. CD4, a co-receptor of the TCR complex, also binds
to a different section of the MHC molecule. With the assistance of different kinases associated to TcR, CD3 and CD4
and a phosphatase present on the intracellular section of
CD45 (common leucocyte antigen), activation of biochemical pathways in the cytosol of the Th cell occurs. These active pathways are known as signal 1 of T cell activation, as
it is the first and primary pro-activation signal in a Th cell.
Following a re-stimulation with same antigen memory T
cells use the same TCR pathways to become re-activated.
The signal 2 step involves verification step that is a protective measure to ensure non-self response, and involves an
interaction between CD28 on the CD4 T cell and the proteins CD80 (B7.1) or CD86 (B7.2) on the professional APCs.
Both CD80 and CD86 activate the CD28 receptor. These proteins are also known as co-stimulatory molecules. Once
both signals 1 and 2 are active within the helper T cell,
the cells are able to proliferate by releasing a potent T
cell growth factor called interleukin-2 (IL-2) that also acts

-glucans in aquaculture
in an autocrine way by binding to up-regulated IL-2 receptors (CD25) on both CD4 and CD8 T cells. This binding,
when a critical number of receptors are triggered, leads
to cell division and cytokine production. After many cell
generations, the Th cell progenitors differentiate into
effector Th cells, memory Th cells, and regulatory Th cells.
Effector Th cells are cells that secrete cytokines, proteins
or peptides that stimulate or interact with other leucocytes, including Th cells.
Effector T cell responses
The T cell response generated (including cytokine
production) may be essential for a successful outcome
from vaccination. Understanding exactly how helper T cells
respond to infection is of major interest in immunology and
such knowledge may be very useful to increase the
effectiveness of vaccination. Proliferating helper T cells
that develop into effector T cells differentiate into two
major subtypes of cells known as Th1 and Th2 cells (also
known as Type 1 and Type 2 helper T cells, respectively).
While it is established that certain cytokine patterns favour
differentiation of Th into Th1 or/and Th2 cells after
activation of nave T cells, it is not fully clear how the
various patterns are decided and how the cytokines
interplay with each other. In a rather schematic and simple
way, Th1 and Th2 responses are facetted by different
cytokine profiles. The presence of relatively high amounts
of interferon gamma (IFN-g) and tumor necrosis factor beta
(TNF-) favours a Th1 response characterised by an increased activity of the cellular arm of defence where killing
efficacy of macrophages and proliferation of CD8 T cells
(CTL) are increased [27]. IFN-g increases the production
of IL-12 by dendritic cells and macrophages in an autocrine
manner, to produce more IFN-g in helper T cells promoting
a stronger Th1 response. IFN-g inhibits IL-4, an important
cytokine within the Th2 response. IL-4, IL-5, IL-6, IL-10
and IL-13 are all involved in a favoured Th2 response that
stimulates B cells into proliferation, antibody class switching and to increase antibody production. Of these interleukins, IL-10 inhibits the transcription of a variety of
cytokines such as IL-2 and IFN-g by all Th cells, and IL-12
in dendritic cells and APC, but continues to stimulate
plasma cells to produce antibodies. While Th1 and Th2 responses are pro-inflammatory, a Th3 response is believed
to be immune suppressive by increased production of IL10 and TGF- of Th1/Th2 cells at a later stage in inflammation [27]. Manifestation of the different Th responses in fish
is still in its infancy due to lack of suitable Th signature antibodies such as antibodies against fish IL-12 and IL-4. However, at a transcriptome level many of these signature
genes could be analysed and quantified.

Vaccines e mode of action

One of the key questions for vaccine development against
infections in fish and higher vertebrates is to understand
the mechanisms underlying the difference between inferior
protection attained by inactivated vaccines and the strong
immunity often induced by replicating variants. Most
inactivated antigens will not induce sufficient immune
response when administered in the absence of an adjuvant.

389
Adjuvanted vaccines typically work through three principal
modes of actions: (1) through increase of antigen uptake
and presentation of antigens (oil emulsion, liposomes,
etc.), (2) by a danger effect (cholera toxin, CpG-ODNs,
LPS), and (3) through signal 2 action (co-stimulation; costimulatory molecules, cytokines, etc.). The use of adjuvants has been largely empirical but today we are seeing
the empirical approach being superseded by a rational
design where one strategy is to deliver an inert antigen in
the context of a danger signal [3]. Oil adjuvants mediate
their danger signal in forms of varying degrees of tissue
damage. In salmon they alarm the immune system likely
by causing tissue damage at the injection and depot site,
the consequence being scarring and adhesions [28,29].
The danger effects may be initiated after ligand binding
by Toll-like receptors.
Some approaches have been made to include immunostimulants as vaccine adjuvants in fish to increase vaccine
potency and efficacy. Increased vaccine potency in terms of
increased antibody response was observed after injection of
-glucan adjuvanted inactivated A. hydrophila vaccine [30].
Although not addressed in this study, the mushroom -glucan
they obtained and applied, resulting in the elevated antibody response, was probably a water-soluble -glucan.
Water-soluble -glucans, often obtained from fungi, have
been thoroughly investigated with respect to biological effects in many mammalian species [31]. Overall, the effects
-glucans exerted include increased mitogenicity, stimulation of hematopoietic stem cells, activation of complement
pathways, and activation of lymphocytes, macrophages,
dendritic cells, NK cells, Th cells, and cytotoxic T cells as
reported in mammalian experimental models. However,
some substances may down-regulate certain inflammatory
responses thus being novel as anti-inflammatory drugs.

-glucans as adjuvants
Modern aquaculture would have been impossible without
the use of injectable vaccines against common diseases.
However, the use of oil emulsions as adjuvants may cause
major drawbacks in terms of unwanted effects such as
internal organ adhesions, melanin deposits, effects on
growth and skeletal deformations. As advocated in
a state-of-the-art review: Although the majority of the
farmed fish seems to possess side effects within acceptable
limits, given the pros of vaccination e it is evident that
some fish possess unacceptable levels of side effects [32].
Adhesions and skeletal deformations are probably the side
effects with the most important implications for animal
welfare and ethics. This addresses the need to develop immunization strategies using milder adjuvants such as glucans or other immunostimulants. Early findings by Rrstad et al. (1993) described protective response of yeast
particulate -glucan adjuvanted A. salmonicida vaccine in
Atlantic salmon [33]. Similar protection was also found applying yeast -glucan in combination with A. salmonicida
bacterin in protection against furunculosis [34]. Following
on in another study, Japanese flounder were fed formalinkilled E. tarda cells together with curdlan (a linear gelling
-glucan), and were assessed for specific antibody response. No increased antibody response was observed

390
although the survival against live E. tarda experimental
challenge was higher than in fish fed the control diet
[35]. This indicated that there was no correlation
between vaccine potency and efficacy. Other defence
mechanisms such as activation of the cellular defence
may have been activated. On the contrary, Midtlyng et al.
(1996) reported no significant protective effects of yeast
-glucan adjuvanted vaccine followed by experimental
challenge with A. salmonicida [36]. Their explanation was
that a high infection pressure in a given challenge experiment was inferior when looking for effects of -glucan adjuvanted vaccines. Following this, absence of adjuvanticity
of yeast -glucan administered with Streptococcus bacterin
to turbot has been reported [37]. Kawakami and co-workers
reported an absence of adjuvant action by using yeast
particulate -glucan co-injected with Pasteurella piscicida
vaccine [38]. Also oral application of yeast -glucan
followed by vaccination with Streptococcus iniae did not
confer protection against S. iniae challenge [10]. Interestingly, zymosan has been found to increase vaccine potency
when co-administered with a HIV-1 DNA vaccine to mouse
[39]. Whether this also applies to fish remains unknown,
but it opens up for applying non-genetic/non-nucleosidic
adjuvants in combination with e.g., VHS and IHN DNA vaccines in aquaculture, especially to DNA vaccines not proven
efficacious enough to induce full protection. Apparently,
due to the conflicting results obtained by -glucans to
increase vaccine potency and efficacy it seems that more
research is highly warranted in terms of dose, duration,
experimental models, and a comprehensive study focusing
on what molecular features (molecular weight, conformation, water-solubility and charge) may be the most potent
one for application to aquaculture systems. In addition,
there may be species-to-species variation with regard to
biological effects, and differential effects against different
pathogens.

-glucan receptors
Non-TLR -glucan receptors
It has been hypothesised that collaboration among
different membrane receptor families, as well as with
non-classical opsonins and protein cascades such as complement, is likely to be an important mechanism to
enhance affinity of binding and specificity [40]. Several
different receptors have been reported to bind -glucans,
these are the scavenger receptors (SR), complement receptor 3 (CD11b/CD18), dectin-1 and TLR2/6. All being innate
receptors but dectin-1 and TLR2/6 are also highly involved
in adaptive responses. There are at least three classes of SR
(SR-A, SR-B and SR-C) based on their structural characteristics. Most of the SR are non-opsonic receptors that display
low-affinity and promiscuous binding to many polyanionic
and modified substances. Although they may bind anionic
-glucans (sulphated -glucans either made chemically or
found in certain algae), they are not considered to be
involved in -glucan binding and internalization. Dectin-1,
belonging to a non-classical C-type lectin-like family that
predominantly binds protein ligands, has been considered
to be the major -glucan receptor [41]. In mouse, dectin1 is expressed on neutrophils, alveolar macrophages, and

R.A. Dalmo, J. Bgwald

inflammatory macrophages, with minor expression on resting macrophages and dendritic cells [40]. It is widely
acknowledged that dectin-1 mediates its own signalling
and induces specific responses such as IL-10 (anti-inflammatory) production in DC and the respiratory burst in macrophages and neutrophils, but it is recently shown that
binding of ligands to dectin-1 alone or together with TLR2
binding of ligands induce other responses to stimuli such
as IFN-g, IL-2 and IL-12p40 production [42]. However,
recent findings have suggested that dectin-mediated fungal
recognition preferentially induces IL-17 producing Th cells
(Th17) in both humans and mice [43]. A Th17 response is important in epithelial cell activation, neutrophil recruitment
and during mucosal homing of leucocytes by modulation of
chemokine receptor expression. The resulting immune
response following infection with extracellular pathogens
and fungi containing dectin-1 ligands may favour Th17
differentiation and it is hypothesised to be important for
eradication of extracellular bacteria and fungi at mucosal
sites. A combined stimulation of both TLR and dectin-1 by
yeast cell wall extract zymosan results in mainly Th1 cell
differentiation. This is probably due to an IL-12 and IFN-g
mediated antagonism of Th17 cell differentiation [43]. The
Th17 differentiation is also dependent on the presence of
transforming growth factor (TGF-), IL-6, and IL-23. The
Th17 response concept would be highly important to address
in fish with use of -glucan as a stimulant to increase mucosal defence activity against bacteria and fungi. In line with
this, five zebrafish IL-17 genes have been cloned and characterised [44], whereas Atlantic salmon IL-17D is currently
being cloned and sequenced (personal communication). In
addition, there are available sequence data on putative
IL-17 gene sequences from rainbow trout (GenBank accession nos. AJ580842 and AJ580843), zebrafish (GenBank accession nos. AB195256, AB195257, AB195258, AB195259 and
AB195260) and lamprey (GenBank accession no. AB303391).
Interestingly, relatively high amount of IL-17 transcripts
were found in mucosal tissues of the zebrafish [44].
-glucan receptors have been found on Atlantic salmon
macrophages [45] and channel catfish neutrophils [46],
whether these belong to non-TLR or TLR remains to be
addressed. Not investigated is the antifungal effect of glucans in fish even though there exists a reproducible
experimental saprolegnia challenge model. Tissue distribution studies on fish have revealed that endothelial cells and
macrophages are mainly responsible for uptake of parenterally injected -glucan, whereas intestinal epithelial cells
(enterocytes) have absorbed -glucan after oral and peranal administration [47]. Any receptor dependent uptake
was not, however, studied, and uptake of particulate yeast
-glucan in intestinal cells of fish has not been proved yet.
Complement receptors
The complement receptor Type 3 (CR3; CD11b/CD18), which
belongs to the family of 2-integrins, is another wellcharacterised -glucan receptor. CR3 has a -glucan binding
site within its {alpha}-chain subunit (CD11b) of the heterodimeric protein [48]. Besides increasing the uptake of iC3b
and C3b opsonised matter facilitating enhanced antigen presentation, activation of CR3 may control IL-12 synthesis of
human monocytes thereby affecting T cell immunity [49].
Functionally, a CR3-dependent rosette formation has been

-glucans in aquaculture
observed in carp [50], while CR3 gene(s) has not been cloned
and fully sequenced yet in any fish species.
Lactosylceramide
Lactosylceramide has been found on leucocytes and endothelial cells, and is a glycolipid containing a hydrophobic
ceramide lipid and a hydrophilic sugar component. It may
bind specifically -glucans with concomitant production of
reactive oxygen metabolites [51,52]. It is not known
whether such binding may affect APC and T cell activities.
Toll-like -glucan receptors
Formerly, it has been widely acknowledged that fungal cells
and zymosan, both consisting of many different molecular
moieties, may bind TLR2 and TLR4 facilitating myD88
dependent intracellular signalling that induce cytokines
favouring Th1 cell differentiation [53]. Zymosan, that is
obtained from yeast cell walls is composed of insoluble glucans and mannan, and have been utilized in numerous
experiments mainly due to its ability to activate macrophages. Lately, it has been questioned that impurities by,
e.g., bacterial lipopolysaccharides may be decisive for activation of TLR4 [52]. To support this, a recent review has
pointed out that the content of mannans, chitins, proteins
and lipids in zymosan may interfere with the interpretation
of results. Lately, the stand-alone activity of dectin-1 and
the cooperation of dectin-1 with TLR2 have been proposed
to be the major and most important events inducing cell
signalling [54].

Gene expression following -glucan

administration
Besides gene expression studies of certain acute phase
products (TNF-a, IL-1, IL-6, complement components 3
and 5 and their subtypes) [55,26,56,19] no targeted
approach has been performed to elicit the effects of
-glucans on adaptive immune responses in fish. In a monocyte cell line (THP-1) a high number of genes were up-regulated following -glucan stimulation, those of particular
interests were genes involved in chemotaxis (chemokine
ligands) such as CXCL1, CXCL2, CXCL3 and Th1 associated
genes such as IL-12, TNF, IL-8 [57]. Taking the Th17 induction capacity by -glucans into consideration together with
the induction of chemokine ligands it may be speculated
that -glucans may have their potential in increasing mucosal disease resistance. The ability of -glucans to increase
the expression of TLR would be important to address since
it may be hypothesised that an up-regulation of, e.g., TLR3,
TLR7 and TLR9 would in turn, when appropriate stimulus is
present, confer increased disease resistance against interferon a/ susceptible viruses, and an up-regulation of
most of the other TLR may increase disease resistance
against intracellular bacteria.

TLR agonists and polarisation of

T cell responses
In response to microbial pathogens, CD4 T cells differentiate
into Th1 and Th2 cells, each of these subsets is responsible

391
for activating immune responses adapted to the type of infectious agent [58]. Th1 cells respond to produce IFN-g that
activate APC, and induce B cells to start production and
release of IgG2 isotype antibodies. The Th1 response is particularly effective against eradication of intracellular pathogens. The other arm, Th2, is supported by production of
IL-4 and IL-5 and may activate humoral components and is
especially protective against parasitic infections. Upon receptor binding TLR agonists may program, already at the
first stage, the APC to favour a Th1 or Th2 response
due to certain intracellular signalling cascades resulting in
gene transcription and cytokine production. The APC express the broadest repertoire of TLR through which they
can recognise and bind a high number of different microbial
products [59]. After TLR ligand binding immature APC (e.g.,
DC) may go through maturation steps and migrate to secondary lymphoid organs concomitant with high MHC and
co-stimulatory expression essential for T cell priming. DCs
may produce, dependent of microbial products or IFN-g
and subsequent CD40L provided by T cells, significant IL12 that push the response towards a Th1 bias [59]. In
most circumstances infectious agents contain an array of
different Toll-like agonists. Most of them induce a Th1
bias, but TLR2 and TLR5 agonists may skew the response
into a Th2. Some of these substances may synergize and increase the potential to trigger nave T cells due to a very
high IL-12 production after binding to DCs. Such high IL-12
(IL-12p70) production has been observed after treating human differentiated monocytes (akin to DC) with a mixture
of LPS (TLR4 agonist) and R848 (TLR7) [60]. Even higher production was observed when IFN-g and CD40L were added.
This effect was temporally sustained even up to 24 h. In
a commercial fish vaccine, including up to six different inactivated pathogens, there are numerous different PAMP
that may serve as PRR agonists that alone or synergistically
can induce certain effects on APC/DCs. What the immunological effects are in terms of APC/DC response together
with their effects on T cell response polarisation is an
open issue for further research. Preliminary results obtained in our laboratory suggest that -glucan (Dectin-1,
TLR2/4 agonist) induces salmon spleen IFN-g gene transcription in vivo supporting a Th1-like response. This was
compared with saline injected controls. Anyhow, more targeted and sophisticated experiments must be performed to
find out whether there are functional Th1, Th2, Treg and
Th17 responses in fish similar to higher vertebrates. Clearly,
more monoclonal antibodies for use in cell sorting/flow cytometry combined with functional in vitro cell assays and
gene expression analysis will solve some of these fundamental issues of innate and adaptive fish immunology.
Importantly, the development of vaccines against intracellular pathogens, being the most challenging disease agents
in aquaculture nowadays, should be based on fundamental
immunology rather than trial and error empirical examination of vaccines.

Immunomodulation in shrimp
b-Glucan has been used to enhance resistance in crustacea
against both viral and bacterial infections. The white spot
syndrome (WSS) is the major disease of shrimp in India,

392
Southeast Asia and the Southern and Central America. The
etiological agent is a DNA virus that consists of at least five
major proteins, of which three are localised to the
nucleocapside and two to the envelope [75]. The WSSV
causes high mortality in many cultured shrimp species including black tiger shrimp (Penaeus monodon) and kuruma
shrimp (Penaeus japonicus). Administration of b-1,3-glucan
has been shown to improve the survival of black tiger
shrimp when challenged with WSSV [61e63]. Post larval
and juvenile shrimps were fed diets containing 2 g kg1
for 10e20 days. Following challenge with WSSV, mortality
was significantly lower in the glucan-fed shrimps. None of
the un-treated shrimps survived longer than 4 days. By contrast, some of the glucan-treated larvae (12%) and some of
the juveniles (20%) were still alive at day 6 and were reared
for another 120 days [61]. In another study P. monodon
were fed diets with different levels of b-1,3-glucan from
Schizophyllum commune and then challenged with injection of WSSV [63]. The survival rate of shrimps fed the
diet containing 10 g kg1 was significantly higher on day 9
than the other groups (0, 1, 2 and 20 g kg1). Haemocyte
phagocytic capability, phenol oxidase activity, superoxide
anion and superoxide dismutase activity were significantly
enhanced after oral b-1,3-glucan administration [63]. Sritunyalucksana et al. [73] investigated both in vivo and in vitro
stimulation of the black tiger prawn by peptidoglycan, LPS
and the b-1,3-glucan laminarin. In this study, laminarin
failed to activate the prophenoloxidase, agglutinin and antibacterial activity. Explanation of this lack of effect is uncertain because b-1,3-glucans have been reported to be
a specific activator of prophenoloxidase in several investigations [73].
Huang and Song injected a b-1,3-1,6-glucan from bakers
yeast (S. cerevisiae) in spawners of P. monodon. Larvae
from these glucan-stimulated spawners seemed to be better protected against challenge with the WSSV. This surprising result is the first indication of a maternal transmission
of immunity in invertebrates [67]. Not well-characterised
methanol extracts of five different herbal medicinal plants
were included in diets for the black tiger shrimp [66]. After
25 days of feeding, the shrimps were challenged intramuscularly with WSSV. The group containing 0.8 g kg1 herbal
extracts in the feed resulted in significantly enhanced
survival. However, the active component in these extracts
is not determined.
Crustaceans have mechanisms to recognise cell wall
components of bacteria and fungi. Examples are b-1,3glucan binding protein (GBP) [72] in the black tiger shrimp
and lipopolysaccharide and b-1,3-glucan (LGBP) in white
shrimp (Litopenaeus vannamei) [64]. The GBP is constitutively expressed in haemocytes and shrimps injected with
a b-1,3-glucan (curdlan) or heat-killed Vibrio harveyi did
not result in altered mRNA expression during the first 12 h
after injection [72]. In the white shrimp, a lipopolysaccharide and b-1,3-glucan binding protein cDNA was cloned from
the haemocyte and hepatopancreas. Expression of this
protein was enhanced in haemocytes 3 and 6 h after Vibrio
alginolyticus injection [64].
Another shrimp species called the kuruma shrimp
(P. japonicus) is also susceptible to the WSSV [70]. In the
study shrimps were intramuscularly (i.m.) injected once
with inactivated WSSV alone or in combination with b-1,

R.A. Dalmo, J. Bgwald

3-glucan and inactivated Vibrio penaeicida. The shrimps
were challenged by i.m. injection of WSSV on the 10th
day post injection. No significant differences in mortality
were observed between the treated group and the control
group [70]. In another trial kuruma shrimps were injected
three times at 10-day intervals with the inactivated WSSV
and in combination with inactivated V. penaeicida. The
shrimps were challenged on the 30th day post-third vaccination. In this trial, shrimps that had been injected with
both WSSV and Vibrio bacteria resulted in a significant
lower mortality [70]. Also, in a brief study by Itoh, P. Japonicus received schizophyllan by the oral route and phagocytic activity and protection against RV-PJ (WSSV)
infection was evaluated [68]. The conclusion of this study
is that an oral dose of schizophyllan provided a protective
response against the RV-PJ infection.
Enhancement of resistance to bacterial infections has
also been demonstrated in shrimps.
A study on white shrimp (L. vannamei) demonstrated
potentiation of survival in shrimps against infection with
V. harveyi fed diets containing S. cerevisiae, Phaffia rhodozyma and the experimental live yeast Saccharomyces
exiguus with the pigment xeaxanthin compared with the
diet containing b-glucan [71].
Formalin-killed V. harveyi bacterin and carboxymethyl
b-1,3-glucan were given to black tiger shrimps by feeding
for 10 days [69]. In another set of experiments, shrimps in
commercial ponds were given pellets containing the two
substances for 2 months. They were then challenged with
virulent V. harveyi. Shrimps in the experimental tanks and
the commercial ponds were better protected against infection after treatment by bacterin, the glucan or combinations thereof [69]. Glucan from spent brewers yeast was
included in the feed and used in the treatment of black
tiger shrimps. In this study it was concluded that phenol
oxidase activity and the number of haemocytes increased
by a factor of 2e3 [74]. Also, in vitro antibacterial activity
of haemolymph against V. harveyi increased significantly.
In a thorough study by Wang et al. (2007) [76] expression
of nine genes important in the immune defence of the
Pacific white shrimp (L. vannamei) was investigated.
Transcripts of all nine genes were detected in all tissues.
b-glucan binding protein-high density lipoprotein (BGBPHDL), lipopolysaccharide-b-glucan binding protein (LGBP)
and haemocyanin were mainly expressed in the hepatopancreas [76]. In a following study the same authors investigated the response to dietary inclusion of a b-1,3-glucan
from S. commune. mRNA expression of BGBP-HDL, LGBP
and cytosolic manganese superoxide dismutase (cMnSOD),
but not haemocyanin was significantly enhanced in the hepatopancreas during a 7-day feeding trial [77]. These molecules play important roles in the activation of the proPO
system, coagulation process and expression of antimicrobial peptides. Also, a temporal up-regulation of mRNA of
lysozyme and cMnSOD expression in haemocytes was found.
Probiotics are live microbial feed supplements that
affect the host by improving the immune function and
resistance against infections. Lactobacillus plantarum is
a probiotic that was used in the feed of the white shrimp
to test for some immune parameters and protection against
infection [65]. The total haemocyte count was significantly
lower in shrimps fed 1010 cfu (kg diet-1) L. plantarum for

-glucans in aquaculture
Table 2

393

Immunostimulant effects in shrimps

Agent

Species

Administration

Effects

Reference

b-1,3-glucan
Schizophyllum
commune
b-1,3-glucan
Schizophyllum
commune
b-1,3-glucan
Schizophyllum
commune
Lactobacillus
probiotics
Immunostimulant
herbs
b-1,3-1,6-glucan
insoluble
(bakers yeast)
Schizophyllan

Black tiger shrimp

(Penaeus monodon)

Oral

Reduced mortality

[61]

Black tiger shrimp

Oral

Enhanced haemocyte phagocytosis, superoxide

anion production, shrimp survival

[62]

Black tiger shrimp

Oral

[63]

White shrimp

Oral

Black tiger shrimp

Oral

Enhanced phenol oxidase, superoxide anion,

superoxide dismutase, survival
(white spot syndrome)
Increased PO and SOD activities.
Increased bacterial clearance
Enhanced survival against white spot syndrome

Black tiger shrimp

Injection/
immersion

Enhanced survival against white spot syndrome

Maternal transfer of immunity

[67]

Kuruma shrimp
(Penaeus japonicus)
Black tiger shrimp

Oral

Protective effect against RV-PJ virus

[68]

Oral

Protection against Vibrio harveyi infection

[69]

Kuruma shrimp
White shrimp
Black tiger shrimp
Black tiger shrimp
Black tiger shrimp

Intramuscular
Oral
Injection
In vitro
Oral

[70]
[71]
[72]
[73]
[74]

White shrimp

Oral

Enhanced protection against white spot syndrome

Reduced resistance
Isolation of a -1,3-glucan binding protein
No effect
Enhanced phenol oxidase, number of haemocytes,
bacterial killing
Enhanced mRNA expression of lysozyme
and superoxide dismutase

Carboxymethyl
b-1,3-glucan
b-1,3-glucan (yeast)
b-1,3-glucan (yeast)
Curdlan, zymosan
Laminarin
Crude b-1,3-glucan
(Brewers yeast)
b-1,3-glucan from
Schizophyllum
commune

48 h. But the PO activity, SOD activity, and the survival after

challenge with V. alginolyticus were all increased. A quite
recent article reports for the first time cloning of a Toll receptor in shrimp (lToll) in the white shrimp [78]. Toll-like receptors are recognising many classes of pathogens important
for the establishment of both innate and adaptive responses.
lToll contains characteristic domains of Toll proteins: an extracellular leucine-rich repeat (LRR) with cysteine-rich motifs and an intracellular domain. Expression studies of lToll
showed occurrence in most tissues like gill, brain, heart,
stomach, intestine, pylorus, muscle, epidermis and haemocytes. Notably, the hepatopancreas showed no or low
expression of the lToll receptors (Table 2).

Conclusion
-glucans have the potential to increase survival rates of
fish and shellfish e at least when applied as a prophylactic
measure where an injection of -glucans may induce
disease resistance. Especially for shellfish, -glucan containing diets may be an interesting option to increase
defence activity thereby disease resistance. One promising
aspect is its potential to activate fish Th17 cells that, in
turn, may increase mucosal disease resistance. The adjuvant effects possessed by some -glucans should be
addressed further. A major challenge is to reveal the

[65]
[66]

[77]

most important innate protective defence mechanisms

that can be modulated by using immunostimulants such as
-glucans.

Acknowledgements
The financial support of the European Commission, grant
IMAQUANIM (contract no. 007103), the NANOMAT program
(contract no. 182035) and RCN FISHVACCPLAT (contract
no. 172508/S40) of the Research Council of Norway is highly
acknowledged.

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