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ROLE OF IMMUNOSTIMULANTS IN IMMUNE

RESPONSES OF FISH AND SHELLFISH

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

52
Immunostimulants1/Rout Immune parameters2 Fish and shellfish Protection against pathogen Reference
AKXA-1/intraperitoneal PA, BA, SOD, Lys Cyprinus carpio Aeromonas hydrophila Wang et al. (2011)
AP (Ep, Ut) +MI (Pp, Cp)/oral THC, PoA, TP, A:G, RBA Litopenaeus vannamei White spot syndrome virus Peraza-Gómez et al. (2014)
Allium sativum Lys, RBA Labeo rohita Aeromonas hydrophila Sahu et al. (2007)
Astragalus embranaceus/oral Lys, PA, RBA Apostichopus japonicas Vibrio splendidus Wang et al. (2009)
Andrographolide/oral Lys, MPO, RBA, PA Labeo rohita Aeromonas hydrophila Basha et al. (2013)
Achyranthes aspera/oral Lys, MPO, TP, A: G, NO Labeo rohita, Cyprinus carpio Aeromonas hydrophila Rao et al. (2006),
Chakrabarti and Srivastava (2012)
Ascorbic acid/ intraperitoneal PA, RBA Mystus gulio Aeromonas hydrophila Anbarasu and Chandran (2001)
AvBA-1, VlBA-2 and FsBA-3/oral ACP, Lys, TP, A:G, RBA, PA Cyprinus carpio Aeromonas hydrophila Chi et al. (2014)
Avicennia marina/oral WBC, Lys, RBA, ACP, PA Amphiprion sebae Vibrio alginolyticus Dhayanithi et al.(2015)
AXOS/oral ACP, RBA, MPO, PA, Igs Geraylou et al. (2013)

P. K. Srivastava and A. K. Pandey

Acipenser baerii ———
Azadirachta indica/oral PA, Lys, TLC Lates calcarifer Vibrio harveyi Talpur and Ikhwanuddin (2013)

Bacillus amyloliquefaciens/oral Lys, NO, IL-1, TNF α Oreochromis niloticus Yersinia ruckeri, Clostridium Selim and Reda (2015)
perfringens
Biofilm of Vibrio alginolyticus /oral THC, PoA, BA Penaeus monodon Vibrio alginolyticus, White spot Sharma et al. (2010b)
syndrome virus
Mannan oligosaccharide (Bio-Mos®)/oral THC, DHC Cherax tenuimanus Vibrio mimicus Sang et al. (2009)
β-glucan/oral or intraperitoneal BA, TLC Oreochromis niloticus Aeromonas hydrophila El -Boshy et al. (2010)
THC, PoA, RBA Fenneropenaeus indicus White spot syndrome virus Sajeevan et al. (2009)
Lys Salmo salar Ichthyobodo necator Paulsen et al. (2001)
THC, DHC Cherax tenuimanus ——— Sang and Fotedar (2010)
Clarias batrachus Aeromonas hydrophila Kumari and Sahoo (2006)
Pseudosciaena croce Vibrio harveyi Ai et al. (2007)
Paralichthys olivaceus Edwardsiella tarda Yoo et al. (2007)
Oncorhynchus mykiss Yersinia ruckeri Skov et al. (2012)
Bacillus simplex DR-834 BA, Lys, PA, RBA, NO Cyprinus carpio Aeromonas hydrophila Wang et al. (2010)
Bovine lactoferrin/oral ACH, TLC, TP, IgM, Lys Acipenser baeri ——— Eslamloo et al. (2012)
Bacillus subtilis T13/oral ACH, NO, PA, RBA, SOD Apostichopus japonicas Vibrio splendidus Zhao et al. (2012)
Bacillus subtilis E20/oral PA, PoA, RBA Litopenaeus vannamei Vibrio alginolyticus Tseng et al. (2009)
Microalgae (Pt + Tc), B. subtilis/oral IgM, PA, RB Sparus aurata Photobacterium damselae Cerezuela et al. (2012)
subsp. piscicida
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 Sparus aurata ——— Cerezuela et al. (2009)
Cholesterol/oral SOD, GSH-Px, CAT, TAC, Oncorhynchus mykiss Aeromonas hydrophila Deng et al. (2013)
ACP, Lys, RBA
Chromium/oral WBC, RBC, Lys, PA Oncorhynchus mykiss ——— Gatta et al. (2001)
Chlorogenic acid/oral TAS, SOD, GSH-Px, CAT, GPx Litopenaeus vannamei ——— Wang et al. (2015a)
Copper/oral TLC, Igs, TP, Lys THC, PoA Puntius gonionotus
Eriocheir sinensis Aeromonas hydrophila Shariff et al. (2001)
Aeromonas hydrophila Sun et al. (2013)
Cyanodon dactylon/intraimuscular or oral THC, NO, PoA Penaeus monodon White spot syndrome virus Balasubramanian et al.(2008)
Cotinus coggygria/oral Lys, MPO, TP, A: GLys, PA, Oncorhynchus mykiss, Vibrio anguillarum Bilen et al. (2011, 2013)
TP, RBA Cyprinus carpio
Coriolus versicolor/Intraperitoneal Lys, MPO, PA, TP, A: G Epinephelus bruneus Listonella anguillarum Harikrishnan et al. (2012a)
Curcumin/oral NF-κB/c-Rel, IL-1β, TNF-α, Cyprinus carpio ——— Cao et al. (2015)
GPx
Daphnia similis/oral PA, RBA Lates calcarifer Aeromonas hydrophila Chiu et al. (2015)
Debaryomyces hansenii CBS 8339/oral CA, PA, ACP, RBA Sparus aurata ——— Reyes-Becerril et al. (2008)
Durio zibethinus/oral THC, PoA Penaeus monodon Vibrio harveyi, White spot Pholdaeng and Pongsamart (2010)
syndrome virus
EcoActivaTM /oral ACP, RBA Pagrus auratus ——— Cook et al. (2001, 2003)
Eclipta alba/oral ACP, Lys, MPO Oreochromis mosambicus Aeromonas hydrophila Christybapita et al. (2007)
Eriobotrya japonica/oral ACP, BA, TL, Lys, PA, TP, A:G Epinephelus bruneus Vibrio carchariae Kim et al. (2011)
Euglena viridis/oral BA, Lys, TP, A: G, RBA Labeo rohita Aeromonas hydrophila Das et al. (2009)
Eichhornia crassipes/oral THC, PoA, RB, GPX, SOD, PA Macrobrachium rosenbergii Lactococcus garvieae Chang et al. (2013a)
Emodin and vitamin C/oral Lys, A: G Megalobrama amblycephala ——— Ming et al. (2012)
Endotoxin/intraperitoneal MPO, RB, RTP, A: G Labeo rohita ——— Nayak et al. (2000)

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

P. K. Srivastava and A. K. Pandey

Gynura bicolor/intraperitoneal THC, RBA, PA, PoA, SOD, Litopenaeus vannamei Vibrio alginolyticus Hsieh et al. (2013)
LGBP
Hanseniaspora opuntiae C21/oral Lys, NO, PoA Apostichopus japonicas Vibtio splendidus Ma et al. (2013)
Hericium erinaceum/oral THC, PoA, RBA Litopenaeus vannamei Vibrio alginolyticus Yeh et al. (2011)
Ixora coccinea, Daemia extensa TLC, TP, A:G Carassius auratus Aeromonas hydrophila Anusha et al. (2014)
and Tridax procumbens/oral
IL-1â/ intraperitoneal Lys Oncorhynchus mykiss Aeromonas salmonicida Hong et al. (2003)
Inulin and vitamin C/oral Lys, RBA Oreochromis niloticus Aeromonas hydrophila Ibrahem et al. (2010)
Inosine monophosphate/oral Lys, MPO, SOD Paralichthys olivaceus Streptococcus iniae Song et al. (2012)
Korean mistletoe/oral Lys, PA, RBA Apostichopus japonicas ——— Choi et al. (2008)
Lycopene/oral TL, PA, TP, A: G, RBA Oncorhynchus mykiss ——— Yonar (2012)
Lslzm protein/intraperitoneal THC, Lys, PA, PoA, RBA Litopenaeus stylirostris White spot syndrome virus Mai and Wang (2010)
Levan/oral Lys, TP, A: G, RBA Cyprinus carpio Aeromonas hydrophila Rairakhwada et al. (2007)
Lactococcus lactis BFE920/oral MPO, TP, A:G Paralichthys olivaceus Streptococcus iniae, Kim et al. (2013)
Streptococcus parauberi,
Enterococcus viikkiensis,
Lactococcus garviae
Lactobacillus plantarum/oral BA Litopenaeus vannamei Vibrio harveyi Khanitta and Tipparat (2012)
Lipopolysaccharide/oral BA, Lys, PA Cyprinus carpio ——— Kadowaki et al. (2013)
Musa acuminate/oral THC, DHC, RBA, SOD, GPx, Macrobrachium rosenbergii Lactococcus garvieae Rattanavichai and Cheng (2015)
PoA, TG
Mushroom â-Glucan /oral 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 Myxocyprinus asiaticus ——— Zhang et al. (2009)
Polyhydroxybutyrate-hydroxyvalerate/ Lys, MPO Oreochromis mossambicus Aeromonas hydrophila Suguna et al. (2013)
oral
Punica granatum/oral Lys, A:G Paralichthys olivaceus Lymphocystis disease virus Harikrishnan et al. (2010)
Pyridoxine/oral PA, PoA, RBA Haliotis discus hannai ——— Chen et al. (2005)
Rheum officinale Lys, TP, A:G Megalobrama amblycephala Aeromonas hydrophila Liu et al. (2012)
Recombinant channel catfish RB, NO, Lys Ictalurus punctatus Aeromonas hydrophila Pridgeon et al. (2013)
lysozyme-g
Rhodotorula benthica D30/oral Lys. NO, PA Apostichopus japonicus ——— Wang et al. (2015b)
Rutin/ /intraperitoneal PO, RBA, SOD Litopenaeus vannamei Vibrio alginolyticus Hsieh et al. (2008)
Ribonucleic acid/oral TLC, TP, A:G, RBA Labeo rohita Aeromonas hydrophila Choudhury et al. (2005)
Reishi immunomodulatory protein (riZ-8) PA, SOD Epinephelus coioides Nervous necrosis virus Kuan et al. (2012)
R pcDNA-GPR18/ intraperitoneal Lys, NO, RBA Ictalurus punctatus Aeromonas hydrophila Pridgeon and Klesius (2013)
Sodium alginate/oral PA, PoA, RBA Litopenaeus vannamei Vibrio alginolyticus Cheng et al. (2004, 2005, 2007)
Epinephelus coicoides, Vibrio parahaemolyticus Cheng and Yu (2013)
Haliotis discus hannai
Sargassum wightii/oral THC, PA, PoA, RBA, SOD Penaeus monodon White spot syndrome virus Immanuel et al. (2012)
Sargassum hemiphyllum/intraperitoneal TLC, THC, POA, PA, Lys Litopenaeus vannamei Vibrio alginolyticus Huynh et al. (2011)
Styrax japonica/oral Ach, MPO, PA, Lys, RBA Epinephelus bruneus Vibrio harveyi, Harikrishnan et al. (2011c)
Uronema marinum
Sophora flavescens/oral ACP, Lys, MPO, RBA Oreochromis niloticus Streptococcus agalactiae Wu et al. (2013)
Sargassum fusiforme polysaccharide THC, TP, Lys, PoA Fenneropenaeus chinensis Vibrio harveyi Huang et al. (2006)
extracts (SFPSE)/oral
Saponin THC, á2-macroglobulin (á-2M), Litopenaeus vannamei Vubrio alginolyticus Su and Chen (2008)

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

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 haemocytes in invertebrates (Franchini et al, 1995),
from the substrate L-arginine to L-citrulline through the directly or indirectly contributing to pathogen elimination.
action of nitric oxide synthases (NOS) (Stefano, 1999). NO can interact directly with tyrosyl radicals to form
This reaction requires cofactors such as NADPH, FAD, nitrotyrosine (indicator or marker of cell damage,
BH4, calcium and calmodulin (Gorren and Mayer, 2007). inflammation as well as NO production) (Davis et al,
In vertebrates, there are three kinds of NOS isoform: 2001) and react indirectly with reactive oxygen species
neuronal NOS (nNOS), endothelial NOS (eNOS) (Ca2+ (ROS, such as hydroxyl radical and superoxide anion) to
/Calmodulin dependent) are constitutively expressed in generate a much more powerful oxidant peroxynitrite
resting cells and inducible NOS (iNOS) (independent of (ONOO -) (Fig. 2) (Thomas et al, 2008). ONOO -
Ca2+) is not present in resting cells and can be mainly substantially increases the toxicity by tyrosine nitration,
activated in monocytes, macrophages, dendritic cells, sulfhydryls modification (Moncada and Bolaños, 2006)
neutrophils and natural killer (NK) cells by cytokines and and poly(ADPribose) polymerase (PARP) ribosylation
endotoxin and mediates the ability of macrophages to kill which is critic for DNA repair and closely related with
or inhibit the growth of bacteria, viruses and fungi (Aktan, apoptosis (Mannick et al, 1996). As an ubiquitous
2004; Yang et al, 2013). The inducible NO synthesised pathogen-killing molecule, NO has been reported to
by iNOS is considered to be involved in the immune mediate the immune response in mollusc whose defense
response, and high amounts of NO are synthesised in the mechanisms rely exclusively on innate immunity including
immunocytes, such as macrophages in mammals and 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 Bachere et al, 1995; Seeley et al, 1990; Chi et al, 2014),
oyster, clam, snail and mussels (Torreilles and Romestand, include particles recognized, bound to the surface of cells
2001; Tafalla et al, 2003; Serfozo et al, 2002; Nieto- and internalized into phagosomes forming around the
Fernandez et al, 2000; Novas et al, 2007). In molluscs, engulfed materials (Stuart and Ezekowitz, 2008). During
NO regulates the metabolic activity of ink gland and pathogen invasion, phagocytosis is the component of the
extent of melanin production (Scheinker et al., 2005). In early innate immune response against infectious agents
crustaceans, NO acts as a cytotoxic molecule contributed and host defiance mechanisms (Stuart and Ezekowitz,
to microorganisms killing (Rodríguez-Ramos et al., 2008; 2008). Pathogens once enters inside, the phagosome are
Yao et al, 2010; Inada et al, 2010) i.e. NO plays a dual destroyed by lowered pH, hydrolysis and radical attack
role in the elicited response as it has both protection in (Henneke and Golenbock, 2004). This process involves
host defence against bacterial or viral pathogens and the recognition and attachment of foreign particles,
cytotoxic actions in some pathological processes (Liew, including pathogens, engulfment and digestion by the
1995). phagocyte i.e it is a form of endocytosis where particles
Phagocytosis (>0.5 mm) are ingested into endocytic vesicles called
phagosomes and initiated by receptor-mediated
Phagocytosis, an evolutionary ancient defense endocytosis or through non-specific hydrophobic
mechanism of innate (non-specific) immunity, are the interactions of the cell membrane with the target particle
first-line cellular defence and conserved in both (Neumann et al, 2001). It is one of the most important
vertebrates and invertebrates (Roth and Kurtz, 2009;
 Immunostimulants in immune responses of fish and shellfish 59
processes in poikilothermic animals because it is the of diphenol to quinones, subsequently leading to the
process that is least influenced by temperature (Lange production of melanin, a microbicidal compound (Lee and
and Magnadottir 2003; Magnadottir et al, 2005). Overland Soderhall, 2002; Cerenius and Soderhall, 2004) i.e. the
et al. (2010) investigated the phagocytic activity of prophenoloxidase cascade has a key role in melanization
neutrophils, as well as the phagocytic ability of B-cells and seems to participate in host defence by augmenting
from the teleost species Atlantic salmon (Salmo salar) phagocytosis, nodule formation, encapsulation and
and Atlantic cod (Gadus morhua). hemocyte locomotion. In addition, proteinase inhibitors
The adaptive immune responses by crustaceans also like pacifastin and α2-macroglobulin (α2-M) play crucial
depend on their innate immune response systems (Lee roles in regulating the proPO-activating system to prevent
and Soderhall, 2002) such as humoral and cellular immune deleterious effects of PO which leads to melanin
systems (Soderhall, 1999). Phagocytosis by haemocytes formation (Soderhall et al, 1986, 1994; Yeh et al, 2009).
is considered to be the main cellular mediated immune ProPO in penaeid shrimp is synthesized and localized in
mechanism of shellûsh. Out of three shrimp hemocytes semi-granular and granular cells (Bayne, 1990; Perazzolo
(hyaline, semi-granular and large granular cells), hyaline and Barracco, 1997; Soderhall and Cerenius, 1998;
and semi-granular cells are responsible for phagocytosis Striunyalucasana et al, 1999a, b) which is activated and
(Giulianini et al, 2007) i.e. In crustaceans, hemocytes converted to phenoloxidase (PO) by a serine proteinase
play important roles in phagocytosis and played an in the presence of a minute amount of microbial
important role in shrimp innate immunity which can be polysaccharides like β-1,3-glucan, lipopolysaccharide
regulated by following protein; white spot syndrome virus (LPS) through pattern recognition molecules or
(WSSV) VP466 protein (Yeh et al, 2012), Rab6 protein endogenous trypsin-like serine proteinase, the so-called
(Wu et al, 2008; Wu and Zhang, 2007; Zong et al, 2008), proPO-activating enzyme (PPA) (Mowat et al, 1991).
Ran protein (Liu et al, 2009), PAP (phagocytosis Different immunostimulant (Table 1) has been reported
activating protein) known as the ribosomal protein L26 to enhance phenoloxidase activity in shrimps.
(RPL26) (Khimmakthong et al, 2011, 2013) against Respiratory burst activity
WSSV infection or activated when there is invasion of Respiratory burst (sometimes called oxidative burst)
any antigen or microorganism. Therefore, hemocytic is the rapid release of reactive oxygen species
phagocytosis is a primary mechanism by which (superoxide radical and hydrogen peroxide) from
hemocytes eliminate and degrade invading pathogens; different types of cells (Fig. 2). Stimulation of the
therefore, levels of phagocytosis largely indicate the phagocytic cell membrane and thereby activation of the
degree of immune cell activation (Mydlarz et al, 2006). membrane associated NADPH oxidase, initiates an
Additionally, phagocytic ability has also been proposed increased oxygen consumption and the production of
as a biomarker for the detection of toxicants in aquatic reactive oxygen species (ROS) which are considered to
toxicology, and it has been used in a tiered system for the be toxic for bacterial pathogens (Sharp and Secombes,
immunotoxicological assessment of environmental 1993; Secombes and Fletcher, 1992; Secombes, 1996;
pollutants and immunostimulants used in aquaculture Hardie et al, 1996; Itou et al, 1996). In crustaceans, the
(Secombes, 1994). Several studied reported that various superoxide anion is a reactive molecule originated during
immunostimulants (Table 1) and bacterial secondary phagocytosis as a product of the hemocytes’ respiratory
metabolites increased the phagocytic capability of cells burst and plays a key role in microbicidal activity (Bell
in fish and shellfish (Dugenci, 2003; Chen et al, 2003; and Smith, 1993), i.e. increased respiratory burst activity
Gopalakannan and Arul, 2006; Namba et al, 2007; Aly et can be correlated with increased bacterial pathogen killing
al, 2008; Wang et al, 2011; Geng et al, 2011; Cerezuela activity of phagocytes or hemocytes which is an important
et al, 2012). indicator of cellular immunity mechanisms in fish and
Phenoloxidase (PO) activity shellfish (Mangum, 1983; Sharp and Secombes, 1993;
In crustaceans, PO activity in hemocytes is the Miyazaki, 1998; Munoz et al, 2000). However, various
terminal enzyme in the prophenoloxidase (proPO) system immunostimulant (Table 1) has been reported that
and can be an indicator to evaluate the health status of significantly enhanced production of intracellular
shrimp (Ratclilfe et al, 1985; Vargas-Albores et al, superoxide anion and widely used to evaluate the defense
1998).The prophenoloxidase (proPO-As) which is ability of a host against different pathogens (Campo-
normally activated by invading microorganisms or Cordova et al, 2002; Cheng et al, 2005; Citarasu et al,
parasites, is a complex enzyme cascade. PO catalyses 2006).
hydroxylation of monophenol to diphenol and oxidation
60 P. K. Srivastava and A. K. Pandey
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