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ROLE OF IMMUNOSTIMULANTS IN IMMUNE

RESPONSES OF FISH AND SHELLFISH

Article · March 2015

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Biochem. Cell. Arch. Vol. 15, No. 1, pp. 47-73, 2015 ISSN 0972-5075

Review Article
ROLE OF IMMUNOSTIMULANTS IN IMMUNE RESPONSES OF FISH AND
SHELLFISH
P. K. Srivastava and A. K. Pandey
National Bureau of Fish Genetic Resources, Canal Ring Road, Lucknow - 226 002, India
e-mail: praveen2ku28@gmail.com
(Accepted 18 March 2015)

ABSTRACT: In recent years, the fish and shellfish consumption has increased but the total world production decreased. The
main cause of the decreased fish production is the occurrence of diseases caused by different bacterial and viral pathogens.
There is a growing need to control, prevent or minimise the devastating effects of diseases in fish and shellfish culture without
recourse to toxic chemicals or antibiotics. In keeping with alternative approaches without using toxic chemicals to control fish
and shellfish diseases, many medicinal plants and their extracts act as immunostimulant mainly by enhancing non-specific
immunity and help in combating the pathogens. These are Cyanodon dactylon, Coriolus versicolor, Eclipta alba, Gracilaria
tenuistipitata, Glucan, Levamisole, Traditional Korean Medicine (TKM), Withania sominiferum, Sargassum hemiphyllum,
Styrax japonica and Tribulus terrestris etc. These immunostimulants mainly facilitate the function of phagocytic cells,
increase myeloperoxidase, bactericidal and lysozyme activities and stimulate the natural killer cells, complement system,
phenoloxidase, respiratory burst activity, nitric oxide synthase and antibody responses which confer enhanced protection from
infectious diseases. Purpose of this review is to summarize and evaluate the current state of knowledge on immunostimulants,
their actions in fish and shellfish immune response and potentials to be applied for enhancing aquaculture production.
Key words : Infectious diseases, natural killer cells, complement system, immunostimulants, immun-parameters, fish, shellfish.

INTRODUCTION worldwide with the top commercial value among species

Fish and shellfish in aquaculture make significant
contributions to the world’s well being and prosperity.
Fish and fishery products are among the most traded food
commodities worldwide, with trade volumes and values
reaching new heights in 2011 and expected to carry on
rising with developing countries continuing to account for
the bulk of world exports. While capture fisheries
production remains stable, aquaculture production keeps
on expanding. Aquaculture is set to remain one of the
fastest-growing animal food-producing sectors and in the
next decade, total production from both capture and
aquaculture will exceed that of beef, pork or poultry. In
the last three decades (1980-2010), world food fish
production of aquaculture has expanded by almost 12
times at an average annual rate of 8.8%. The growth
rate in farmed food fish production from 1980 to 2010
far outpaced that for the world population (1.5%),
resulting in average annual per capita consumption of
farmed fish rising by almost seven times. In crustacean
aquaculture, specifically in penaeid shrimp, the production
of this species has expanded considerably during the
1980s and now represents a multi-billion dollar industry.
White shrimp, Litopenaeus vannamei is the most
important cultivated species of penaeid shrimp in the
of fish, crustaceans and molluscs in global aquaculture
production. The global annual production of freshwater
prawns in 2010 was about 670,000 tons, of which China
produced 615,000 tons (92%). (Mohney et al, 1994;
Flegel, 1997; FAO, 2011, 2012).
The global fish and shellfish aquaculture has been
consistently embosomed by diseases that cause severe
losses in production are major problems that can lead to
umpteenth economic losses. The main contributors to
these losses are due to different bacterial, viral, fungi
and protozoa pathogens like white spot syndrome virus
(WSSV), Vibrio spp., Yersinia ruckeri, Streptococcus
iniae, Aeromonas hydrophila, Edwardsiella tarda
Streptococcus agalactiae (Alavandi et al, 2004; Qi et
al, 2009; Yuan-Hwa et al, 2010; Zhao et al, 2012; Giri et
al, 2012; Park and Jeong, 1996; Song et al, 2012; Wu et
al, 2013). Aquaculture is under pressure to decrease the
use of synthetic antibiotics and chemotherapeutics
because of the risks posed to humans by chemical
residues in foods and by antibiotic resistance being passed
on to human pathogens which threaten the safety and
hygiene of food and control of diseases in humans.
However, the use of antibiotics in aquaculture has been
restricted in many countries (Rijkers et al, 1980; Hsieh
48 P. K. Srivastava and A. K. Pandey
et al, 2013). Therefore, alternative strategies are required,
efforts are being made to exploit plants, plant extracts,
and natural plant compounds as potential alternatives to
synthetic chemicals to stimulate immune responses and
disease resistance (Chakraborty and Hancz, 2011;
Kadowaki et al, 2013; Wu et al, 2013). Fish and shellfish,
unlike vertebrates, are believed to lack adaptive immunity
and completely depend on their innate immunity (Arala-
Chaves et al, 2000; Esteban et al, 2005; Little et al,
2005). Therefore, products which can enhance host
immunity and disease resistance such as
immunostimulants are being used in disease prevention
and have garnered much interest in recent years (Sakai,
1999). Immunostimulants comprise a group of biological
and synthetic compounds that improved both specific
(antibody and agglutination titre) and non-specific
immunity (lysozyme, complement, myeloperoxidase,
phagocytic, bactericidal activity, respiratory burst activity,
nitric oxide, total haemocytes, phenoloxidase) against the
infectious diseases in different fish and shellfish species
(Robertsen et al, 1994; Anderson and Siwycki, 1995;
Amar et al, 2004; Ai et al, 2006; Andrzej et al, 2006;
Behera et al, 2011; Kaleeswaran et al, 2011; Safarpour
et al, 2011; Chakrabarti et al, 2012; Srivastava and
Chakrabarti, 2012; Ambas et al, 2013). Natural
immunostimulants are biocompatible, biodegradable, cost-
effective, safe for the environment as well as human
health and enhancing disease and stress resistance
(Kadowaki et al, 2013; Ortuno et al, 2002). Many natural
compounds from various sources (bacterial components,
chemical agents, animal or plant extracts etc.) have been
investigated as prospective immunostimulants against
pathogen infection in aquaculture. Various
immunostimulants which has been scrutinized to enhance
the immune response in fish and shellfish constitutes
different active principal compound like saponin,
glycyrrhizin, aloe, azadirachtin and essential fatty acids
(EFAs) Linolenic and oleic acids (Ninomiya et al, 1995;
Jang et al, 1995; Kim et al, 1999; Harikrishnan et al,
2009; Chakrabarti et al, 2012). A large number of herbal
plants and other immunuostimulants have been used in
traditional medicine for the treatment and the control of
several diseases in aquaculture, these are dimerized
lysozyme (Siwicki et al, 1998), Solanum trilobatum
(Divyagnaneswari et al, 2007), β-endorphin (Watanuki
et al, 2000), CpG oligodeoxynucleotides (Tassakka and
Sakai, 2002, 2003; Meng et al, 2003), Isin (Villamil et al,
2003), recombinant transferring (Stafford et al, 2004),
bovine lactoferrin (Esteban et al, 2005), Spirulina
platensis (Watanuki et al, 2006), Eclipta alba
(Christybapita et al, 2007), Astragalus membranaceus
and Lonicera japonica (Ardo et al, 2008), Euglena
viridis (Das et al, 2009), Toona sinensis (Wu et al, 2010),
Sargassum hemiphyllum (Huynh et al, 2011),
Aachyranthes aspera (Chakrabarti and Srivastava,
2012), Urtica dioica (Binaii et al, 2013) and Tribulus
terrestris (Gultepe et al, 2014).
The innate and adaptive immune responses
The innate system is the earliest immune mechanism
that defends the host from infection by other organisms
in a non-specific manner. This means that the cells of
the innate system recognize and respond to pathogens in
a generic way. It also possesses memory as the host
evolves its innate immune components based on
evolutionary experience of its ancestors encountering
similar pathogens (Kurtz, 2005; Diaz-Rosales et al, 2006;
Das et al, 2011). In fish and shellfish, the innate response
has been considered as essential component in combating
pathogens due to limitations of the adaptive immune
system, their poikilothermic nature, their limited repertoire
of antibodies and the slow proliferation, maturation and
memory of their lymphocytes (Dunier, 1994; Whyte,
2007). The innate immune system is initiated and triggered
by foreign polysaccharide particles, known as pathogen-
associated molecular patterns (PAMPs), that are
recognized and bound by pattern recognition proteins
(PRPs) and these in turn activate phagocytosis and the
prophenoloxidase (proPO) activating system (Janeway
and Medzhitov, 2002; Cerenius et al, 2008). Shrimp and
other invertebrates have developed an innate immune
system in which haemocytes play a crucial role in
defending against hostile microorganisms for self-
protection (Loker et al, 2004; Kitikiew et al, 2013). Three
types of haemocytes (hyaline, semigranular and granular
cells) are recognized based on their cell size and degree
of granularity (Tsing et al, 1989; Zhang et al, 2006).
Acquired/adaptive/specific immunity plays a vital role
in protection against recurrent infections by generating
memory cells (cell-mediated immunity) and specific
soluble and membrane-bound receptors (humoral
immunity) such as T-cell receptors and immunoglobulin
(Ig) which allow the fast and efficient elimination of the
specific fish pathogens (Ellis, 2001; Swain, 2006).
Immunoglobulin (antibodies) expressed either as B
lymphocyte receptor or secreted in plasma. Effectively
only one functional immunoglobulin class, a tetrameric
IgM, is present in teleosts. This is in contrast to the five
immunoglobulin classes and several sub-classes of
mammals. Other Ig-like molecules have been described
in some fish species which may increase the diversity of
the B-cell recognition capacity (Wilson et al, 1997). The
lymphocytes the B- and T- cells, are responsible for
specific pathogen recognition and initiation of the adaptive
 Immunostimulants in immune responses of fish and shellfish
immune response. The B-cells are involved in the humoral
response while the T-cells are responsible for the cell
mediated response (Randelli et al, 2008). Major immune
responses which are mostly enhanced by
immunostimulants include antimicrobial peptides,
Hemmaglutination titre, Igs levels, lysozyme,
myeloperoxidase activity, respiratory burst activity,
alternate complement activity, nitric oxide synthase, serum
bactericidal activity and total serum protein, albumin,
globulin. haemograms, phenoloxidase (PO) activity,
phagocytic activity, and clearance efficiency are
considered common markers to evaluate a fish and
shrimp’s health (Rodriguez and Moullac, 2000).
Alternate complement activity
Complement is an essential part of the innate immune
system and involves about 35 soluble and membrane-
bound proteins. The functions of complement are
numerous but it is most well known for its capacity to kill
pathogens by creating pores in their surface membranes
(Holland et al, 2002; Gasque, 2004). Complement-
mediated killing occurs when complement is activated
either directly by microorganisms or by antibody-antigen
(Ag-Ig) complexes. This activation by Ag-Ig complexes
makes complement an important effectors mechanism
for the adaptive immune response. One fascinating aspect
of complement is its role in modulating the adaptive
immune response by binding to specific receptors on
mammalian lymphocyte surfaces and follicular dendritic
cells (Fearon and Locksley, 1996; Carroll and Prodeus,
1998; Sahu and Lambris, 2001). Complement thereby
provides an important link between adaptive and innate
immune responses. Therefore, evolution of the
complement system acts as a bridge between innate and
adaptive immunity which first evolved in fish (Sunyer et
al, 2003; Dunkelberger and Song, 2010) i.e. Complement
is a system of serum proteins, which is central to many
defense mechanisms and plays an essential role in alerting
the host of the presence of potential pathogens as well
as in their clearing (Boshra and Sunyer, 2006). It contains
three pathways: the classical, alternative, and lectin
pathways (Fig. 1) (Carroll, 2004; Hawlisch and Kohl,
2006; Zhou et al, 2012). Spontaneous haemolytic activity,
attributed to the alternative complement pathway, is
commonly high in fish serum (Lange et al, 2001). Both
marine and freshwater fish have the alternative
complement pathway and classical complement pathway
of activation, directly comparable to those of mammals
(Holland et al, 2002). The alternative complement activity,
which is antibody independent, is very prominent in fish
serum compared to mammalian serum suggesting that
this pathway is more important in fish than mammals. It
49
is one of the three complement pathways that opsonize
and kill pathogens. The end result of activating the
classical, alternative or lectin pathways is production of
an active C5 convertase. The terminal sequences interact
sequentially to form a macromolecular structure called
the membrane-attack complex (MAC) which will result
into their lysis (Fig. 1). The bactericidal activity of
complement has been reported in many fishes (Boshra
and Sunyer, 2006) and has been well recognized as one
of the key killing mechanism of clearing bacteria in teleost
(Leiro et al, 1996; Jenkins and Ourth, 1993) which can
be activated by different immunostimulants (Table 1).
Antimicrobial peptides (AMPs)
AMPs are distributed in most living organisms as a
key component of the innate immune system ranging from
bacteria to plants and animals (Mygind et al, 2005). These
peptides exhibit a wide range of activities against
numerous microbes including bacteria, fungi, viruses and
parasites while with little or no toxicity to host cells
(Hancock and Scott, 2000). Although fish are the most
diversified group of vertebrates till now, relatively few
AMPs have been isolated from them which reflected
presumably a simple lack of attention to this potentially
rich source of AMPs. The antibacterial peptides have
been isolated from a number of fish species e.g. sole fish
(Pardachirus marmoratrus - Lazarovici and Loew,
1986), common carp (Cyprinus carpio - Lemaitre et al,
1996), white bass x striped bass (Morone chrysops x
M. saxatilis - Shike et al, 2002), tilapia (Oreochomis
massambicus - Chen et al, 2009) and catfish (Mystus
gulio and Arius maculatus - Anbuchezhian et al, 2011)
and crabs (Carcinus maenas- Schnapp et al, 1996;
Callinectes sapidus - Khoo et al, 1999) and also from
the shrimp (Penaeus vannamei - Destoumieux et al,
1997). Such peptide antibiotics seem to be important
defence molecules in all living organisms including
bacteria, plants, invertebrates and vertebrates (Boman,
1995). Increasing numbers of antibacterial peptides have
been identified from teleosts in recent years and some
can be activated against microorganisms (Cuesta et al,
2008; Maier et al, 2008; Chia et al, 2010). Isolation and
characterization of a novel antibacterial peptide derived
from hemoglobin α in the liver of Japanese eel, Anguilla
japonica (AJHbα), the first identified fragment of
haemoglobin α chain showed strong antibacterial activity
in fish (Zhang et al, 2013). Antony et al (2011) identified
molecular characterization of a crustin-like antimicrobial
peptide in the giant tiger shrimp (Penaeus monodon),
and its expression profile in response to various
immunostimulants and challenge with white spot
syndrome virus (WSSV). In most of the crustacean
50 P. K. Srivastava and A. K. Pandey
species, the antimicrobial activity has been located in the
haemolymph and/or in the haemocytes. However, potent
antimicrobial activity has also been detected in other
organs/tissues (Mori and Stewart, 1978; Stabili et al, 1999;
Jayasankar and Subramoniam, 1999).
Bactericidal activity
Bactericidal activity is possibly correlated with
phagocytosis and killing activity of neutrophils,
macrophages and is involved in eradication of pathogenic
microbes in fish (Ellis, 1999). Antimicrobial activity has
been detected in several decapods crustaceans including
lobster, crabs, shrimps and freshwater crayfish (Schwab
et al, 1966; Stewart and Zwicker, 1972; Chisholm and
Smith, 1992; Noga et al, 1996; Carrington and Secombes,
2007). However, little is known about the nature of the
antimicrobial factors in crustaceans and only a few
compounds have been fully characterized (Haug et al,
2002). Chattopadhyay et al. (1996) isolated an
antimicrobial lectin, named scyllin, from the mud crab
(Scylla serrata). Increased serum bactericidal activities
indicate that various humoral factors involved in innate
and/or adaptive immunities are elevated in the serum to
protect the host effectively from infection (Wang et al,
2010). The higher bactericidal activities can possibly be
due to a higher production of O2-. It was reported that
higher levels of bactericidal activity occurred in fish and
shellfish after treating with different immunostimulant
(Table 1) (Chen and Anisworth, 1992; Smith et al, 1995;
Sahu et al, 2007; Das et al, 2009; Kim et al, 2011; Ji et
al, 2012).
Immunoglobulins (Igs) and Hemmaglutination/
Aglutination titre levels
The immunoglobulins (Igs or antibodies), which are
the principal indicators of acquired immunity to pathogens
have evolved toward producing highly diversified
molecules that recognize a remarkably large number of
different antigens. As a natural antibody, IgM is the main
immunoglobulin present in teleosts (Ellis, 1998) and could
provide an instant protection of ûsh against pathogens.
Therefore, its reflect the immune status of fish
(Magnadottir, 2010; Israelsson et al, 1991). Fish IgM are
usually tetrameric as opposed to the pentameric structure
of mammalian IgM. The occurrence of fish IgM is not
limited to the serum. Antibodies are also found in the fish
epithelial mucus and these IgM may be of local origin
rather than derived from the serum (Lobb and Clem, 1981;
Peleteiro and Richards, 1985) which suggested that fish
immune system could be viewed as consisting of systemic
and mucosal part (Ellis, 1988). The hemagglutination
assay is a method of quantification for viruses or bacteria
by hemagglutination. Antigen antibody response is can
be measure by Hemagglutination antibody (HA) titre
levels (Chakrabarti et al, 2012). A natural factor such as
agglutination may help to overcome diseases earlier than
that required to produce specific immunity (Sahoo et al,
2005). Natural fish haemagglutinins/agglutinins bind to
carbohydrate moieties of microbes, causing agglutination
for inactivation and easy immune clearance (Dalmo et
al, 1997). Several humoural elements of the non-specific
immune system are present in plasma such as
immunoglobulins, transferrin, agglutinins and precipitins
(Magnadóttir, 2006). Due to agglutinin’s role as opsonins
in the process of phagocytosis by the host haemocytes,
agglutination titre and phagocytic ratio have a theoretically
strong relationship (Ordas et al, 2000; Olafsen et al,
1992). These elements significant increases at
supplementation of various immunostimulants (Table 1).
Leucocytes/haemocyte count
The ability of leucocytes to kill pathogenic microbes
is probably one of the most important protection
mechanisms (Miyazaki, 1998). Leucocytes/ haemocytes
play an important role in non-specific or innate immunity
and their count can be considered as an indicator of the
health status of fish (Sritunyalucksana et al, 1999; Le
Moullac and Haffner, 2000; Flores-Miranda et al, 2011).
Many works has been done to show that the herbal plant
act as immunostimulants (Table 1) which significantly
influenced leucocyte/haemocyte count and activated
white blood cells (WBC) such as macrophages,
granulocytes and monocytes responsible for defence
against infections and supports the repair of damaged
tissues. (Zhang, 1994; Sahoo and Mukherjee, 1999; Jian
and Wu, 2003; 2004; Choudhary et al, 2005; Misra et al,
2006; Sahu et al, 2007; Harikrishnan et al, 2010; Shiau
and Jiang, 2006; Nya and Austin, 2009; Abdel-Tawwab
et al, 2010). Monitoring of WBC profile can reflect
general immune system status in fish. Fish leucocytes
have been classified using criteria that primarily apply to
mammalian counterparts (Ellis, 1977) and several studies
have suggested that many fish leucocytes show
morphological resemblance and functional similarities with
mammalian cells (Zinkl et al, 1991; Groff and Zinkl, 1999;
Rowley, 1990). A hemocyte is a cell found in the
hemolymph and plays a role in the immune system of
invertebrates and they are responsible for adhesion,
phagocytosis, nodule formation, encapsulation,
melanization, cytotoxicity, and hemolymph clotting
mechanism (Soderhall and Cerenius, 1992; Sequeira et
al, 1996; Song and Hsieh, 1994; Lin et al, 2006; Cerenius
et al, 2008). Classification of the haemocyte types in
decapod crustaceans is based mainly on the presence of
 Immunostimulants in immune responses of fish and shellfish
cytoplasmic granules into hyaline, semigranular and
granular cells (Johansson et al, 2000).
Lysozyme
Lysozyme is a cationic protein that is present in mucus,
lymphoid tissue, plasma as well as in other fluids and is
also expressed in a wide variety of tissues (Saurab and
Sahoo, 2008). In fish, lysozyme is synthesized in both
liver and extra hepatic sites (Bayne and Gerwick, 2001)
and involved in a broad range of defence mechanisms
such as bacteriolysis, opsonisation, immune response,
antimicrobial as well as restricted antiviral and
antineoplastic activity as found in higher vertebrates
(Chipman and Sharon, 1969; Krogdahl et al, 2000; Losso
et al, 2000; Magnodottir, 2006; Saurab and Sahoo, 2008;
Maqsood et al, 2010). It acts on the peptidoglycan layer
of bacterial cell walls resulting in the lysis of bond between
N-acetylmuramic acid and N-acetylgluco-samine
(Hjelmeland et al, 1983).
Lysozyme is thought to provide protection against
adenovirus and could be used to cure bleb, parotitis,
chicken pox, hepatitis, flu, blocked HIV-1 replication in
infected cells and its derived peptides exhibit DNA-
binding activity, which may interfere with DNA
replication, modulate gene expression and block bacterial
and viral infections, contributing to the proteins’ innate
immune modulating activity (Huang et al, 1999; Lu and
Wang, 2005; Lin et al, 2009). It is most studied innate
response in fish and shellfish which is significantly
enhanced by various immunostimulants (Table 1) (Engstad
and Robertson, 1993; Thompson et al, 1995). In zebra
fish, lysozyme genes are expressed in cells of myeloid
origin and are very early in development (Hall et al, 2007).
Lysosomal hydrolytic enzymes have been scarcely studied
in crustaceans, but as in molluscs, the hemocytes of
shrimp produce several such enzymes (Carajaraville et
al, 1995; Peraza-Gómez et al, 2011). The lysozymes of
penaeid are well characterized and shown to have lytic
activity against several species of Gram positive and Gram
negative bacteria, including pathogenic species of Vibrio
(de la Re-Vega et al, 2006; Hikima et al, 2003).
Myeloperoxidase activity (MPO)
Neutrophils are an important component of host
defence against many bacterial, viral and fungal infections
and the evaluation of neutrophil function is valuable for
assessment of the health status of individuals and animal
populations (Densen and Madell, 1990). Myeloperoxidase
is most abundantly expressed lysosomal protein stored in
azurophilic granules neutrophil, measure neutrophil
antimicrobial activity specifically primary granule
exocytosis (Menegazzi et al, 1992; Quade and Roth, 1997;
51
Swain et al, 2007). Measuring exocytosis of
myeloperoxidase from primary neutrophil granules in-vitro
is a direct, rapid and quantitative method to assess the
degranulation process in neutrophils (Menegazzi et al,
1992). MPO is an important enzyme for many fish species
(Castro et al, 2008) and release of meloperoxidase by
neutrophils and monocytes during inflammation play an
important role in innate immune response because it
enhances neutrophil (Lau et al, 2005) and macrophages
(Grattendick et al, 2002) in the blood. The enhancement
of MPO in fish fed with different immunostimulant
supplements have been reported (Table 1). Most studies
document that fish neutrophils have very similar
histochemical staining properties to mammalian
neutrophils and can be distinguished by the presence of
myeloperoxidase in their cytoplasmic granules (Zinkl et
al, 1991; Afonso et al, 1998; Palic et al, 2005).
Granulocyte characterisation in cyprinid species (Cyprinis
carpio, Carassius auratus) describes the presence of
myeloperoxidase in fish neutrophils (Zinkl et al, 1991;
Groff and Zinkl, 1999). It produces hypochlorous acid
from hydrogen peroxide and chloride ion during the
neutrophil’s respiratory burst (Dalmo et al, 1997).
Furthermore, it oxidizes tyrosine to tyrosyl radical using
hydrogen peroxide as an oxidizing agent. Hypochlorous
acid and tyrosyl radical are cytotoxic, so they are used
by the neutrophil to kill bacteria and other pathogens (Fig.
2).
Nitric oxide synthase
Nitric oxide (NO) is an important ubiquitous gaseous
signaling molecule which involved in a broad range of
disease pathogenesis, defeat parasites and considerably
reduce the infectious rate in many species (Lim et al,
2005; Villamil et al, 2007; Bogdan et al, 2000; Bogdan,
2001; Rivero, 2006; AlFadhli et al, 2011; Steinert et al,
2010). It also help in physiological processes including
vascular regulation (Godecke et al, 2001; Figueroa et al,
2002; Guarino et al, 2004; Paduch and Kandefer-
Szerszen, 2009), neural transmission (Steinert et al, 2010;
Schweighofer and Ferriol, 2000; Straub et al, 2007) and
due to high oxidative capability plays an indispensable
role in immunity of all vertebrates and invertebrates (Foley
and O’Farrell, 2003; Nappi et al, 2000; Chakravortty
and Hensel, 2003; Bogdan, 2001; Sharma et al, 2010a)
which can be augmented by some immunostimulant
(Table. 1). In aquatic invertebrates, biological roles of
NO were found to be related to feeding, immune defence,
environmental stress, learning, metamorphosis, swimming,
symbiosis, haemocyte aggregation and regulation of blood
pressure (Palumbo, 2005). It is produced by a variety of
cells such as neurons, astrocytes, immunocytes, and
Table 1 : List of immunostimulants and their effect on different immune parameters against pathoges in fish and shellfish.
Immunostimulants1/Rout Immune parameters2
AKXA-1/intraperitoneal PA, BA, SOD, Lys
AP (Ep, Ut) +MI (Pp, Cp)/oral THC, PoA, TP, A:G, RBA
Allium sativum Lys, RBA
Astragalus embranaceus/oral Lys, PA, RBA
Andrographolide/oral Lys, MPO, RBA, PA
Achyranthes aspera/oral Lys, MPO, TP, A: G, NO
Ascorbic acid/ intraperitoneal PA, RBA
AvBA-1, VlBA-2 and FsBA-3/oral ACP, Lys, TP, A:G, RBA, PA
Avicennia marina/oral WBC, Lys, RBA, ACP, PA
AXOS/oral ACP, RBA, MPO, PA, Igs
Azadirachta indica/oral PA, Lys, TLC
Bacillus amyloliquefaciens/oral Lys, NO, IL-1, TNF α Oreochromis niloticus
Biofilm of Vibrio alginolyticus /oral THC, PoA, BA
Mannan oligosaccharide (Bio-Mos®)/oral THC, DHC
β-glucan/oral or intraperitoneal BA, TLC
THC, PoA, RBA
Lys
THC, DHC
Bacillus simplex DR-834 BA, Lys, PA, RBA, NO
Bovine lactoferrin/oral ACH, TLC, TP, IgM, Lys
Bacillus subtilis T13/oral ACH, NO, PA, RBA, SOD
Bacillus subtilis E20/oral PA, PoA, RBA
Microalgae (Pt + Tc), B. subtilis/oral IgM, PA, RB
Fish and shellfish
Cyprinus carpio
Litopenaeus vannamei
Labeo rohita
Apostichopus japonicas
Labeo rohita
Labeo rohita, Cyprinus carpio
Mystus gulio
Cyprinus carpio
Amphiprion sebae
Acipenser baerii
Lates calcarifer
Penaeus monodon
Cherax tenuimanus
Oreochromis niloticus
Fenneropenaeus indicus
Salmo salar
Cherax tenuimanus
Clarias batrachus
Pseudosciaena croce
Paralichthys olivaceus
Oncorhynchus mykiss
Cyprinus carpio
Acipenser baeri
Apostichopus japonicas
Litopenaeus vannamei
Sparus aurata
Protection against pathogen
Aeromonas hydrophila
White spot syndrome virus
Aeromonas hydrophila
Vibrio splendidus
Aeromonas hydrophila
Aeromonas hydrophila
Aeromonas hydrophila
Aeromonas hydrophila
Vibrio alginolyticus
———
Vibrio harveyi
Yersinia ruckeri, Clostridium
perfringens
Vibrio alginolyticus, White spot
syndrome virus
Vibrio mimicus
Aeromonas hydrophila
White spot syndrome virus
Ichthyobodo necator
———
Aeromonas hydrophila
Vibrio harveyi
Edwardsiella tarda
Yersinia ruckeri
Aeromonas hydrophila
———
Vibrio splendidus
Vibrio alginolyticus
Photobacterium damselae
subsp. piscicida
52
Reference
Wang et al. (2011)
Peraza-Gómez et al. (2014)
Sahu et al. (2007)
Wang et al. (2009)
Basha et al. (2013)
Rao et al. (2006),
Chakrabarti and Srivastava (2012)
Anbarasu and Chandran (2001)
Chi et al. (2014)
Dhayanithi et al.(2015)
Geraylou et al. (2013)
P. K. Srivastava and A. K. Pandey
Talpur and Ikhwanuddin (2013)
Selim and Reda (2015)
Sharma et al. (2010b)
Sang et al. (2009)
El -Boshy et al. (2010)
Sajeevan et al. (2009)
Paulsen et al. (2001)
Sang and Fotedar (2010)
Kumari and Sahoo (2006)
Ai et al. (2007)
Yoo et al. (2007)
Skov et al. (2012)
Wang et al. (2010)
Eslamloo et al. (2012)
Zhao et al. (2012)
Tseng et al. (2009)
Cerezuela et al. (2012)
Table 1 continued...
Table 1 continued...
Camellia sinensis/oral BA, TP, SOD Oncorhynchus keta, Ichthyobodo necator, Suzuki et al. (2006), Abdel-Tawwab
Oncorhynchus masou, Aeromonas hydrophila, et al. (2010), Sheikhzadeh et al. (2011),
Oreochromis niloticus Yersinia ruckeri Nootash et al. (2013)
Oncorhynchus mykiss
Cedrus deodara/oral ACP, Antiprotease, TP, BA, IgM Sparus aurata _____________ Awad et al. (2015)
Chitosan and Bacillus subtilis/oral Ach, Lys, PA, RBA Rachycentron canadum Vibrio harveyi Geng et al. (2011)
Cinnamomum kanehirae/intraperitoneal PoA, PA, RBA Litopenaeus vannamei Vibrio alginolyticus, Vibrio Yeh et al. (2009)
alginolyticus
Chitooligosaccharides/oral TLC, DLC, Lys, PA, RBA Trachinotus ovatus Vibrio harveyi Lin et al. (2012)

Immunostimulants in immune responses of fish and shellfish

Cholecalciferol (D3)/oral ACP, MPO, PA, RBA
Cholesterol/oral SOD, GSH-Px, CAT, TAC,
ACP, Lys, RBA
Chromium/oral WBC, RBC, Lys, PA
Chlorogenic acid/oral TAS, SOD, GSH-Px, CAT, GPx
Copper/oral TLC, Igs, TP, Lys THC, PoA
Aeromonas hydrophila Sun et al. (2013)
Cyanodon dactylon/intraimuscular or oral THC, NO, PoA
Cotinus coggygria/oral Lys, MPO, TP, A: GLys, PA,
TP, RBA
Coriolus versicolor/Intraperitoneal Lys, MPO, PA, TP, A: G
Curcumin/oral NF-κB/c-Rel, IL-1β, TNF-α,
GPx
Daphnia similis/oral PA, RBA
Debaryomyces hansenii CBS 8339/oral CA, PA, ACP, RBA
Durio zibethinus/oral THC, PoA
EcoActivaTM /oral ACP, RBA
Eclipta alba/oral ACP, Lys, MPO
Eriobotrya japonica/oral ACP, BA, TL, Lys, PA, TP, A:G Epinephelus bruneus
Euglena viridis/oral BA, Lys, TP, A: G, RBA
Eichhornia crassipes/oral THC, PoA, RB, GPX, SOD, PA Macrobrachium rosenbergii
Emodin and vitamin C/oral Lys, A: G
Endotoxin/intraperitoneal MPO, RB, RTP, A: G
Sparus aurata ———
Oncorhynchus mykiss Aeromonas hydrophila
Oncorhynchus mykiss ———
Litopenaeus vannamei ———
Puntius gonionotus
Eriocheir sinensis Aeromonas hydrophila
Penaeus monodon White spot syndrome virus
Oncorhynchus mykiss, Vibrio anguillarum
Cyprinus carpio
Epinephelus bruneus Listonella anguillarum
Cyprinus carpio ———
Lates calcarifer Aeromonas hydrophila
Sparus aurata ———
Penaeus monodon Vibrio harveyi, White spot
syndrome virus
Pagrus auratus ———
Oreochromis mosambicus Aeromonas hydrophila
Vibrio carchariae
Labeo rohita Aeromonas hydrophila
Lactococcus garvieae
Megalobrama amblycephala ———
Labeo rohita ———
Cerezuela et al. (2009)
Deng et al. (2013)
Gatta et al. (2001)
Wang et al. (2015a)
Shariff et al. (2001)
Balasubramanian et al.(2008)
Bilen et al. (2011, 2013)
Harikrishnan et al. (2012a)
Cao et al. (2015)
Chiu et al. (2015)
Reyes-Becerril et al. (2008)
Pholdaeng and Pongsamart (2010)
Cook et al. (2001, 2003)
Christybapita et al. (2007)
Kim et al. (2011)
Das et al. (2009)
Chang et al. (2013a)
Ming et al. (2012)
Nayak et al. (2000)

53
Table 1 continued...

Table 1 continued...
Eriobotrya japonica /oral
Ficus carica/oral
Fructooligosaccharide/oral
Fucoidan/oral
Ferritin/intravenous
Gracilaria tenuistipitata/oral,
intraperitoneal
Green tea/oral
Gelidium amansii/Intraperitoneal
Garlic/oral
ACH, HA, PA, TP, Lys
Lys, BA, IL-1â, TNF-á, HSP70
ACH, Lys
TLC, RBC, Hb, Lys
THC, PA, PoA, RBA
PoA, TP, A:G
THC, PO, RBA, SOD
THC, PO, RBA, SOD, Lys
ACP
THC, PA, PoA, RBA
TLC,
RBA
54
Epinephelus bruneus Vibrio carchariae Kim et al. (2011)
Ctenopharyngodon idella ——— Yang et al. (2015)
Rutilus rutilus, ——— Soleimani et al. (2012)
Acipenser stellatus ——— Akrami et al. (2013)
Penaeus monodon White spot syndrome virus Immanuel et al. (2012)
Litopenaeus vannamei Vibrio alginolyticus Kitikiew et al. (2013)
Litopenaeus vannamei White spot syndrome virus Ruan et al. (2010)
Litopenaeus vannamei Vibrio alginolyticus, Hou and Chen (2005), Yeh and Chen
White spot syndrome virus (2009), Yeh et al. (2010),
Sirirustananun et al. (2011)
Epinephelus bruneus Vibrio carchariae Harikrishnan et al. (2011)
Litopenaeus vanname Vibrio alginolyticus Fu et al. (2007)
Clarias gariepinus Aeromonas hydrophila Thanikachalam et al. (2010)
Oncorhynchus mykiss Aeromonas hydrophila Nya and Austin (2011)

P. K. Srivastava and A. K. Pandey

Gynura bicolor/intraperitoneal
Hanseniaspora opuntiae C21/oral
Hericium erinaceum/oral
Ixora coccinea, Daemia extensa
and Tridax procumbens/oral
IL-1â/ intraperitoneal
Inulin and vitamin C/oral
Inosine monophosphate/oral
Korean mistletoe/oral
Lycopene/oral
Lslzm protein/intraperitoneal
Levan/oral
Lactococcus lactis BFE920/oral
Lactobacillus plantarum/oral
Lipopolysaccharide/oral
Musa acuminate/oral
Mushroom â-Glucan /oral
THC, RBA, PA, PoA, SOD, Litopenaeus vannamei Vibrio alginolyticus Hsieh et al. (2013)
LGBP
Lys, NO, PoA Apostichopus japonicas Vibtio splendidus Ma et al. (2013)
THC, PoA, RBA Litopenaeus vannamei Vibrio alginolyticus Yeh et al. (2011)
TLC, TP, A:G Carassius auratus Aeromonas hydrophila Anusha et al. (2014)
Lys Oncorhynchus mykiss Aeromonas salmonicida Hong et al. (2003)
Lys, RBA Oreochromis niloticus Aeromonas hydrophila Ibrahem et al. (2010)
Lys, MPO, SOD Paralichthys olivaceus Streptococcus iniae Song et al. (2012)
Lys, PA, RBA Apostichopus japonicas ——— Choi et al. (2008)
TL, PA, TP, A: G, RBA Oncorhynchus mykiss ——— Yonar (2012)
THC, Lys, PA, PoA, RBA Litopenaeus stylirostris White spot syndrome virus Mai and Wang (2010)
Lys, TP, A: G, RBA Cyprinus carpio Aeromonas hydrophila Rairakhwada et al. (2007)
MPO, TP, A:G Paralichthys olivaceus Streptococcus iniae, Kim et al. (2013)
Streptococcus parauberi,
Enterococcus viikkiensis,
Lactococcus garviae
BA Litopenaeus vannamei Vibrio harveyi Khanitta and Tipparat (2012)
BA, Lys, PA Cyprinus carpio ——— Kadowaki et al. (2013)
THC, DHC, RBA, SOD, GPx, Macrobrachium rosenbergii Lactococcus garvieae Rattanavichai and Cheng (2015)
PoA, TG
ACP, Lys, PA, PoA Epinephelus coioides Vibrio alginolyticus Chang et al. (2013b)
Table 1 continued...
Table 1 continued...
Macogard (0.1%) and Ergosan (0.5%)/oral ACP, Lys Dicentrarchus labrax ——— Bagni et al. (2005)
Navicula/oral ACP, MPO, PA Sparus aurata ——— Reyes-Becerril et al. (2013)
Organic selenium/oral THC Cherax cainii Vibrio mimicus Nugroho and Fotedar (2013)
PLGA-OMP/intraperitoneal TL, MPO Labeo rohita Aeromonas hydrophila Behera et al. (2010)
Pediococcus acidilactici/oral ACP, HA, Igs, Lys, Aequidens rivulatus ——— Neissi et al. (2013)
Phellinus linteus/oral ACP, Lys, PA, SOD Epinephelus bruneus Vibriosis Harikrishnan et al. (2011)
Pseudomonas aeruginosa VSG-2/oral ACP, Lys, PA, RBA, SOD Labeo rohita Aeromonas hydrophila Giri et al. (2012)
Propolis/oral TL, PA, TP, Igs, RBA Oncorhynchus mykiss ——— Yonar et al. (2011)

Immunostimulants in immune responses of fish and shellfish

Propolis and Herba Epimedii extracts/oral Lys, PA, RBA
Polyhydroxybutyrate-hydroxyvalerate/
oral
Punica granatum/oral
Pyridoxine/oral
Rheum officinale
Recombinant channel catfish
lysozyme-g
Rhodotorula benthica D30/oral
Rutin/ /intraperitoneal
Ribonucleic acid/oral
Reishi immunomodulatory protein (riZ-8) PA, SOD
R pcDNA-GPR18/ intraperitoneal
Sodium alginate/oral
Sargassum wightii/oral
Sargassum hemiphyllum/intraperitoneal
Styrax japonica/oral
Sophora flavescens/oral
Sargassum fusiforme polysaccharide
extracts (SFPSE)/oral
Saponin
Myxocyprinus asiaticus ——— Zhang et al. (2009)
Lys, MPO Oreochromis mossambicus Aeromonas hydrophila Suguna et al. (2013)
Lys, A:G Paralichthys olivaceus Lymphocystis disease virus Harikrishnan et al. (2010)
PA, PoA, RBA Haliotis discus hannai ——— Chen et al. (2005)
Lys, TP, A:G Megalobrama amblycephala Aeromonas hydrophila Liu et al. (2012)
RB, NO, Lys Ictalurus punctatus Aeromonas hydrophila Pridgeon et al. (2013)
Lys. NO, PA Apostichopus japonicus ——— Wang et al. (2015b)
PO, RBA, SOD Litopenaeus vannamei Vibrio alginolyticus Hsieh et al. (2008)
TLC, TP, A:G, RBA Labeo rohita Aeromonas hydrophila Choudhury et al. (2005)
Epinephelus coioides Nervous necrosis virus Kuan et al. (2012)
Lys, NO, RBA Ictalurus punctatus Aeromonas hydrophila Pridgeon and Klesius (2013)
PA, PoA, RBA Litopenaeus vannamei Vibrio alginolyticus Cheng et al. (2004, 2005, 2007)
Epinephelus coicoides, Vibrio parahaemolyticus Cheng and Yu (2013)
Haliotis discus hannai
THC, PA, PoA, RBA, SOD Penaeus monodon White spot syndrome virus Immanuel et al. (2012)
TLC, THC, POA, PA, Lys Litopenaeus vannamei Vibrio alginolyticus Huynh et al. (2011)
Ach, MPO, PA, Lys, RBA Epinephelus bruneus Vibrio harveyi, Harikrishnan et al. (2011c)
Uronema marinum
ACP, Lys, MPO, RBA Oreochromis niloticus Streptococcus agalactiae Wu et al. (2013)
THC, TP, Lys, PoA Fenneropenaeus chinensis Vibrio harveyi Huang et al. (2006)
THC, á2-macroglobulin (á-2M), Litopenaeus vannamei Vubrio alginolyticus Su and Chen (2008)

55
Table 1 continued...

Table 1 continued...
SOD, PA, RBA
Spirulina platensis/oral THC, Lys, PoA, RBA
PA
Sulfated galactans/oral THC, PoA
Tribulus terrestris/oral Lys, MPO, TP, A:G
Traditional Chinese Medicine /oral Lys, RBA, PA, THC, PA
Tinospora cordifolia/intraperitoneal ACP, Lys, MPO, RBA, NO
Toona sinensis/intraperitoneal Lys, PA, RBA
Tuftsin/intraperitoneal ACP, BA, TLC, Lys, PA
Litopenaeus vannamei
Cyprinus carpio
Gracilaria fisheri
Oreochromis niloticus
Pseudosciaena crocea,
Cyprinus carpio, Haliotis
discus hannai, Myxocyprinus
asiaticus
Oreochromis mosambicus
Oreochromis mosambicus
Labeo rohita
Vibrio alginolyticus
Edwardsiella ictaluri
White spot syndrome virus
———
Vibrio alginolyticus
Aeromonas hydrophila
Aeromonas hydrophila
Aeromonas hydrophila,
Edwardsiella tarda
56
Tayag et al. (2010)
Watanuki et al. (2006)
Wongprasert et al. (2013)
Gultepe et al. (2014)
Jian and Wu (2003, 2004),
Xue et al. (2008)
Alexander et al. (2010)
Wu et al. (2010)
Misra et al. (2006)

P. K. Srivastava and A. K. Pandey

Traditional Korean Medicine/oral ACP, Lys, PA, RBA Paralichthys olivaceus ——— Harikrishnan et al. (2010)
Tryptophan and methionine/oral BA, ACP Dicentrarchus labrax ——— Machado et al. (2015)
Urtica dioica/oral TLC, Lys, TP, A: G, RBA Huso huso ——— Binaii et al. (2013)
Urtica dioica/oral RBC, WBC, Htc, MCH, Labeo victorianus Aeromonas hydrophila Charles et al. (2015)
MCHC, Np
Vitamin E/oral Lys, TLC, THC, RBA, SOD Labeo rohita Edwardsiella tarda Sahoo and Mukherjee (2002),
Penaeus monodon ——— Lee and Shiau (2004)
Withania somnifera/oral THC, PoA, BA, NO, SOD, Igs, Macrobrachium rosenbergii, Aeromonas hydrophila Harikrishnan et al. (2012b),
Lys, RBA Labeo rohita Sharma et al. (2010c)
Yeast extract, Brewer’s Yeast, TLC, RBA Labeo rohita Aeromonas hydrophila Andrews et al. (2011)
Spirulina/oral
Zingerone/oral THC, Lys, PA, PoA Litopenaeus vannamei Vibrio alginolyticus Chang et al. (2012)
Zingiber officinale/oral BA, Lys, PA, RBA Oncorhynchus mykiss Aeromonas hydrophila Nya and Austin (2009)
Zooshikella sp./oral Lys, PA, RBA Paralichthys olivaceus Streptococcus iniae Kim et al. (2010)
1
(immunostimulant): AKXA-1, Anoxybacillus kamchatkensis XA-1; AP (Ep, Ut) +MI (Pp, Cp), Antiviral plants (Echinacea purpurea and Uncaria tomentosa) + Microbial immunostimulants
(Pediococcus parvulus and Candida parapsilosis); AvBA-1, VlBA-2 and FsBA-3, Aeromonas veronii BA-1, Vibrio lentus BA-2, and Flavobacterium sasangense BA-3; AXOS, Arabinoxylan-
oligosaccharides; microalgae (Pt and Tc) and Bacillus subtilis, (Phaeodactylum tricornutum and Tetraselmis chuii) and Bacillus subtilis; PLGA-OMP, Poly D, L-lactide-co-glycolic acid- outer membrane
proteins of A. hydrophila, O. mossambicus; R pcDNA-GPR18, Recombinant pcDNA - G-protein coupled receptor 18 (GPR18).
2
(Immune parameter): ACP, Alternative complement; BA, Bactericidal activity; Igs, Immunoglobulin; HA, Hemagglutination titre; TLC, Total leucocyte count, THC, Total haemocyte
count; DHC, Differential haemocyte count; SOD, Superoxide dismutase; GSH-Px, Glutathione-peroxidase; GPx, Glutathione peroxidase, CAT, Catalase; TAC, Total antioxidant capacity;
Lys, Lysozyme activity; MPO, myeloperoxidae; NO, Nitric oxide; PA, Phagocytic activity; PoA, phenoloxidase activity; TP, Total protein; A:G, Albumin/ Globulin; RBA, Respiratory burst
activity; SOD, Super oxide dismutase; LGBP, Lipopolysaccharide- and â-1,3-glucan-binding protein; TG, Transglutaminase activity; RBC, Red blood cell, WBC, White blood cell; Htc,
Haematocrit; MCH, Mean cell haemoglobin; MCHC, Mean cell haemoglobin concentration; Np, Netrophiles; IL-1â, Interleukin 1-â; TNF-á, Tumor Necrosis Factor á; HSP70, Heat shock
protein 70; TAS, Total antioxidant status.
 Immunostimulants in immune responses of fish and shellfish 57

Classical Lectin Alternate

C3

C3b

Inflammation

Phagocytosis C5

MAC

Cell lysis

Fig. 1 : The Complement system is a central component of the innate immune system and plays a pivotal role in host defense against
infection. Three arms of the Complement system-the Classical (antigen-antibody complexes), Lectin and Alternative pathways
(Antibody-Independent). The terminal sequences of complement activation interact sequentially to form a macromolecular structure
called the membrane-attack complex (MAC) which results in cell lysis. The Complement system can exert its effects by directly
destroying pathogens by facilitating their removal via phagocytosis, and by alerting and activating other immune cells to sites of
infection and inflammation.

endothelial cells in both vertebrates and invertebrates
from the substrate L-arginine to L-citrulline through the
action of nitric oxide synthases (NOS) (Stefano, 1999).
This reaction requires cofactors such as NADPH, FAD,
BH4, calcium and calmodulin (Gorren and Mayer, 2007).
In vertebrates, there are three kinds of NOS isoform:
neuronal NOS (nNOS), endothelial NOS (eNOS) (Ca2+
/Calmodulin dependent) are constitutively expressed in
resting cells and inducible NOS (iNOS) (independent of
Ca2+) is not present in resting cells and can be mainly
activated in monocytes, macrophages, dendritic cells,
neutrophils and natural killer (NK) cells by cytokines and
endotoxin and mediates the ability of macrophages to kill
or inhibit the growth of bacteria, viruses and fungi (Aktan,
2004; Yang et al, 2013). The inducible NO synthesised
by iNOS is considered to be involved in the immune
response, and high amounts of NO are synthesised in the
immunocytes, such as macrophages in mammals and
haemocytes in invertebrates (Franchini et al, 1995),
directly or indirectly contributing to pathogen elimination.
NO can interact directly with tyrosyl radicals to form
nitrotyrosine (indicator or marker of cell damage,
inflammation as well as NO production) (Davis et al,
2001) and react indirectly with reactive oxygen species
(ROS, such as hydroxyl radical and superoxide anion) to
generate a much more powerful oxidant peroxynitrite
(ONOO -) (Fig. 2) (Thomas et al, 2008). ONOO -
substantially increases the toxicity by tyrosine nitration,
sulfhydryls modification (Moncada and Bolaños, 2006)
and poly(ADPribose) polymerase (PARP) ribosylation
which is critic for DNA repair and closely related with
apoptosis (Mannick et al, 1996). As an ubiquitous
pathogen-killing molecule, NO has been reported to
mediate the immune response in mollusc whose defense
mechanisms rely exclusively on innate immunity including
cellular and humoral immunity. NO and NOS has been
58 P. K. Srivastava and A. K. Pandey
Tyrosine
RBA
Tyrosyl
H2O2 radical
ROS MPO

Superoxide anion H2O2 Cl- HOCl

NO
L-arginine
Peroxynitrite NOS Nitrotyrosine

L-citrullin
Caspase activation Lipid peroxidation Indicator or marker of cell
Protein oxidation damage, inflammation as
Protein nitration well as NO production
Inactivation of enzyme
Apoptosis
Necrosis

Fig. 2 : Innate immune response present in fish and shellfish: Respiratory burst activity (RBA) is the rapid release of reactive oxygen species (O2-
and H2O2) from different types of cells. Usually it denotes the release of these chemicals from immune cells, e.g., neutrophils and monocytes,
as they come into contact with different pathogens. Myeloperoxydase (MPO) produces hypochlorous acid (HOCl) from hydrogen
peroxide (H2O2) and chloride anion (Cl-) during the neutrophil’s respiratory burst. Furthermore, it oxidizes tyrosine to tyrosyl radical using
hydrogen peroxide as an oxidizing agent. HOCl and tyrosyl radical are cytotoxic, so they are used by the neutrophil to kill bacteria and other
pathogens. NO can interact directly with tyrosyl radicals to form nitrotyrosine. It is an indicator or marker of cell damage, inflammation as
well as NO production. It is ifs formed in the presence of the active metabolite NO. Generally in many disease states, oxidative stress
increases the production of superoxide (O2-) and NO forming ONOO- (peroxy nitrite, which could lead to the formation of pro-xidant
peroxynitrite (ONOO-). The production of ONOO- is capable of oxidizing several lipoproteins and of nitrating tyrosine residues in many
proteins. Formation of peroxynitrite in vivo has been ascribed to the reaction of the free radical superoxide with the free radical nitric oxide.
Reactions of peroxynitrite leading to either apoptotic or necrotic cell death.

detected in the haemocytes of several molluscs including
oyster, clam, snail and mussels (Torreilles and Romestand,
2001; Tafalla et al, 2003; Serfozo et al, 2002; Nieto-
Fernandez et al, 2000; Novas et al, 2007). In molluscs,
NO regulates the metabolic activity of ink gland and
extent of melanin production (Scheinker et al., 2005). In
crustaceans, NO acts as a cytotoxic molecule contributed
to microorganisms killing (Rodríguez-Ramos et al., 2008;
Yao et al, 2010; Inada et al, 2010) i.e. NO plays a dual
role in the elicited response as it has both protection in
host defence against bacterial or viral pathogens and
cytotoxic actions in some pathological processes (Liew,
1995).
Phagocytosis
Phagocytosis, an evolutionary ancient defense
mechanism of innate (non-specific) immunity, are the
first-line cellular defence and conserved in both
vertebrates and invertebrates (Roth and Kurtz, 2009;
Bachere et al, 1995; Seeley et al, 1990; Chi et al, 2014),
include particles recognized, bound to the surface of cells
and internalized into phagosomes forming around the
engulfed materials (Stuart and Ezekowitz, 2008). During
pathogen invasion, phagocytosis is the component of the
early innate immune response against infectious agents
and host defiance mechanisms (Stuart and Ezekowitz,
2008). Pathogens once enters inside, the phagosome are
destroyed by lowered pH, hydrolysis and radical attack
(Henneke and Golenbock, 2004). This process involves
the recognition and attachment of foreign particles,
including pathogens, engulfment and digestion by the
phagocyte i.e it is a form of endocytosis where particles
(>0.5 mm) are ingested into endocytic vesicles called
phagosomes and initiated by receptor-mediated
endocytosis or through non-specific hydrophobic
interactions of the cell membrane with the target particle
(Neumann et al, 2001). It is one of the most important
 Immunostimulants in immune responses of fish and shellfish
processes in poikilothermic animals because it is the
process that is least influenced by temperature (Lange
and Magnadottir 2003; Magnadottir et al, 2005). Overland
et al. (2010) investigated the phagocytic activity of
neutrophils, as well as the phagocytic ability of B-cells
from the teleost species Atlantic salmon (Salmo salar)
and Atlantic cod (Gadus morhua).
The adaptive immune responses by crustaceans also
depend on their innate immune response systems (Lee
and Soderhall, 2002) such as humoral and cellular immune
systems (Soderhall, 1999). Phagocytosis by haemocytes
is considered to be the main cellular mediated immune
mechanism of shellûsh. Out of three shrimp hemocytes
(hyaline, semi-granular and large granular cells), hyaline
and semi-granular cells are responsible for phagocytosis
(Giulianini et al, 2007) i.e. In crustaceans, hemocytes
play important roles in phagocytosis and played an
important role in shrimp innate immunity which can be
regulated by following protein; white spot syndrome virus
(WSSV) VP466 protein (Yeh et al, 2012), Rab6 protein
(Wu et al, 2008; Wu and Zhang, 2007; Zong et al, 2008),
Ran protein (Liu et al, 2009), PAP (phagocytosis
activating protein) known as the ribosomal protein L26
(RPL26) (Khimmakthong et al, 2011, 2013) against
WSSV infection or activated when there is invasion of
any antigen or microorganism. Therefore, hemocytic
phagocytosis is a primary mechanism by which
hemocytes eliminate and degrade invading pathogens;
therefore, levels of phagocytosis largely indicate the
degree of immune cell activation (Mydlarz et al, 2006).
Additionally, phagocytic ability has also been proposed
as a biomarker for the detection of toxicants in aquatic
toxicology, and it has been used in a tiered system for the
immunotoxicological assessment of environmental
pollutants and immunostimulants used in aquaculture
(Secombes, 1994). Several studied reported that various
immunostimulants (Table 1) and bacterial secondary
metabolites increased the phagocytic capability of cells
in fish and shellfish (Dugenci, 2003; Chen et al, 2003;
Gopalakannan and Arul, 2006; Namba et al, 2007; Aly et
al, 2008; Wang et al, 2011; Geng et al, 2011; Cerezuela
et al, 2012).
Phenoloxidase (PO) activity
In crustaceans, PO activity in hemocytes is the
terminal enzyme in the prophenoloxidase (proPO) system
and can be an indicator to evaluate the health status of
shrimp (Ratclilfe et al, 1985; Vargas-Albores et al,
1998).The prophenoloxidase (proPO-As) which is
normally activated by invading microorganisms or
parasites, is a complex enzyme cascade. PO catalyses
hydroxylation of monophenol to diphenol and oxidation
59
of diphenol to quinones, subsequently leading to the
production of melanin, a microbicidal compound (Lee and
Soderhall, 2002; Cerenius and Soderhall, 2004) i.e. the
prophenoloxidase cascade has a key role in melanization
and seems to participate in host defence by augmenting
phagocytosis, nodule formation, encapsulation and
hemocyte locomotion. In addition, proteinase inhibitors
like pacifastin and α2-macroglobulin (α2-M) play crucial
roles in regulating the proPO-activating system to prevent
deleterious effects of PO which leads to melanin
formation (Soderhall et al, 1986, 1994; Yeh et al, 2009).
ProPO in penaeid shrimp is synthesized and localized in
semi-granular and granular cells (Bayne, 1990; Perazzolo
and Barracco, 1997; Soderhall and Cerenius, 1998;
Striunyalucasana et al, 1999a, b) which is activated and
converted to phenoloxidase (PO) by a serine proteinase
in the presence of a minute amount of microbial
polysaccharides like β-1,3-glucan, lipopolysaccharide
(LPS) through pattern recognition molecules or
endogenous trypsin-like serine proteinase, the so-called
proPO-activating enzyme (PPA) (Mowat et al, 1991).
Different immunostimulant (Table 1) has been reported
to enhance phenoloxidase activity in shrimps.
Respiratory burst activity
Respiratory burst (sometimes called oxidative burst)
is the rapid release of reactive oxygen species
(superoxide radical and hydrogen peroxide) from
different types of cells (Fig. 2). Stimulation of the
phagocytic cell membrane and thereby activation of the
membrane associated NADPH oxidase, initiates an
increased oxygen consumption and the production of
reactive oxygen species (ROS) which are considered to
be toxic for bacterial pathogens (Sharp and Secombes,
1993; Secombes and Fletcher, 1992; Secombes, 1996;
Hardie et al, 1996; Itou et al, 1996). In crustaceans, the
superoxide anion is a reactive molecule originated during
phagocytosis as a product of the hemocytes’ respiratory
burst and plays a key role in microbicidal activity (Bell
and Smith, 1993), i.e. increased respiratory burst activity
can be correlated with increased bacterial pathogen killing
activity of phagocytes or hemocytes which is an important
indicator of cellular immunity mechanisms in fish and
shellfish (Mangum, 1983; Sharp and Secombes, 1993;
Miyazaki, 1998; Munoz et al, 2000). However, various
immunostimulant (Table 1) has been reported that
significantly enhanced production of intracellular
superoxide anion and widely used to evaluate the defense
ability of a host against different pathogens (Campo-
Cordova et al, 2002; Cheng et al, 2005; Citarasu et al,
2006).
60 P. K. Srivastava and A. K. Pandey
Total protein, albumin and globulin
Proteins are the most important compounds in the
serum and tissue with albumin and globulin being the major
serum proteins. Serum or tissue proteins, albumin, and
globulin play a signiûcant role in immune response and
its increase thought to be associated with a stronger innate
response and considered as basic index of fish and
shellfish health (Mulcahy, 1971; Wiegertjes et al, 1996;
Downs et al, 2001; Campa-Cordova et al, 2005; Jha et
al, 2007; Magnadottir, 2010; Alexander et al, 2011;
Srivastava and Chakrabarti, 2012). Albumin is tested to
check how well the liver and kidneys are working whereas
globulin is tested to determine chances of developing an
infection and consists of several components of which
three groups are easily identified as α-, β- and γ-globulin.
The γ-globulin fraction is the source of almost all the
immunoglobulins of blood gamma (Kumar et al, 2005;
Misra et al, 2006). Different immunostimulants has been
reported to enhanced protein levels in fish and shellfish
(Table. 1).
Conclusion and future perspectives
In this review, the benefits of immunostimulants for
aquaculture and its role in immune responses mainly non-
specific were discussed. The innate immune system is
the primary defence weapon of invertebrates. In fish and
shellfish the innate immune response is not only a
fundamental defence mechanism but also plays an
instructive role in the acquired immune response and
homeostasis. The complex mechanism of non-specific
stimulation and interacting with the specific immune
system to render enhanced protection needs to be further
explored. Their efficacies, particularly to combat parasitic
or viral disease conditions, are worth studying. The
characteristics of immunostimulants are shown in different
tables. It is well known that various immunostimulants
used in aquaculture experiments induce beneficial effects
such as disease protection due to augmentation, cellular
and humoral responses. Immunostimulants enhances
immunocompetence and disease resistance in cultured
fish and shellfish and also have additional advantages,
such as growth enhancement and increased survival rates
under stress. It may be used as an alternative to the drugs,
chemicals and antibiotics currently used to control fish
and shellfish diseases in culture. Thus it is attracting the
attention of many researchers. A comprehensive
research effort should be diverted to study their efficacy
on large-scale field applications and the research efforts
should also be directed to characterize the receptors on
the target cells for recognition of immunostimulatory
substances.
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