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Biotechnological Advances in Shrimp Health Management in the Philippines, 2015: 45-71

ISBN: 978-81-308-0558-0 Editors: Christopher Marlowe A. Caipang, Mary Beth I. Bacano-Maningas
and Fernand F. Fagutao

3. Use of immunostimulants in shrimp

culture: An update
Mary Jane S. Apines-Amar1 and Edgar C. Amar2
1
Institute of Aquaculture, College of Fisheries and Ocean Sciences, University of the Philippines
Visayas, Miagao, Iloilo, Philippines; 2Aquaculture Department,
Southeast Asian Fisheries Development Center, Tigbauan, Iloilo, Philippines

Abstract. Different approaches are used to prevent and control

diseases in aquaculture. Immunostimulation is one method that is
gaining popularity and is considered a promising development in
aquaculture. Immunostimulants were found to be effective in
enhancing parameters of non-specific immunity and resistance to
diseases of fish and crustaceans. However, some issues raised on the
use of immunostimulants pertains to the short-term nature of
immune indices used during efficacy evaluation, possible
detrimental effects during long-term administration, or self-damage
due to unregulated production of immune effectors. Further testing
in large-scale production units has been recommended. This chapter
presents the various types and sources of immunostimulants
commonly used in aquaculture and in shrimp culture in particular.
The effects of each immunostimulant vary depending on its source,
dose, route of administration, length of exposure, and the species to
which it is administered.

Correspondence/Reprint request: Dr. Mary Jane S. Apines-Amar, Institute of Aquaculture, College of Fisheries
and Ocean Sciences, University of the Philippines, Visayas, Miagao, Iloilo, Philippines
E-mail: [email protected]
46 Mary Jane S. Apines-Amar & Edgar C. Amar

Introduction
Shrimp farming has been practiced in many countries for decades.
However, the increasing global demand for shrimp has led to the
intensification of shrimp culture that is one of the major contributory factors
in the emergence of infectious diseases. The causative agents of infectious
diseases in shrimp are mainly viruses and bacteria. Vibrio spp. contribute to
a number of epizootic diseases causing serious problems in shrimp culture
and are responsible for shrimp mass mortalities (Nash et al 1992).
Vaccination in fish and immunostimulation both in shrimp and fish for
disease management are being increasingly appreciated in the aquaculture
industry. To fight against diseases, fish larvae and aquatic invertebrates rely
on their innate immune system (Kurtz & Franz 2003; Little & Kraaijeveld
2004) consisting of cellular and humoral components that cooperate to
recognize and eliminate foreign microorganisms and pathogens (Bachère
2003).
Approaches employed to prevent/control fish diseases include the use
of vaccines, chemotherapeutics, and immunostimulants. Immunostimulants
and vaccines are used as prophylactic interventions. They are not
recommended for use when disease is already present in the host.
Chemotherapeutants, on the other hand, are effective for teatment of
diseases that has already occurred, but they are often subject to abuse and
leave residues that affect product quality, among other issues. Vaccination
is the most reliable method, however, there are no effective vaccines
against most viral diseases. In crustaceans, vaccination is the subject of
ongoing investigations but an efficacious product is not expected to be
available for commercial application in the immediate future.
Immunostimulants may compensate for the limitations of
chemotherapeutants and vaccines. They have modulatory effects on the
immunocompetence and disease resistance of fish and crustaceans. When
combined with vaccination, immunostimulation may also increase the
potency of vaccines. Thus, the use of immunostimulants for health
management is a promising new development in shrimp aquaculture
(Jadhav et al 2006; Barman et al 2013).

The shrimp immune system

The immune system of shrimp is relatively primitive/less well-
developed when compared to that of fish. Invertebrates such as shrimp lack
an antibody-based vertebrate-type adaptive immune system. Thus, non-specific
Use of immunostimulants in shrimp culture: An update 47

immune responses are considered to play crucial roles in the shrimp defence
against pathogens. The induction of innate or non-specific immunity is
therefore a potentially better tool to combat pathogens present in the culture
system compared to vaccination, for example. Phagocytosis, encapsulation,
nodule formation, cytotoxicity, lectins, antimicrobial proteins or peptides,
and prophenoloxidase (proPO), have been confirmed to perform essential
roles in the immune defence of shrimps (Soderhall & Cerenius 1992;
Iwanaga & Lee 2005; Amar & Almendras 2010). In recent years, many
studies conducted in crustaceans and insects provide evidence supporting the
existence of specificity and memory mediated by highly variable receptors in
the immune system in these taxonomic groups (Rowley & Powell, 2007).
However, this phenomenon was not found to be universal in the crustacean
species studied and appears to be pathogen- and host-specific. Moreover, it
is not clear at the moment how this specific immune response can provide
practical benefits for health improvement in shrimp aquaculture.

What are immunostimulants?

Immunostimulants are substances that activate the immune system of
animals to make them more resistant to microbial infections (Raa 1996). The
definition has been expanded somewhat to include live organisms or their
products that have an impact on the immune system. The use of
immunostimulants does not generate a specific response to a certain antigen,
but causes an overall response that hastens recognition and elimination of a
broad range of infectious agents and foreign substances (Campos et al 1993;
Secombes 1994; Sordello et al 1997). Two categories of conserved molecular
patterns are recognized by the innate immunity through the various patttern
recognition receptors (PRRs): (1) non-self or pathogen-associated molecules;
and (2) molecules generated as a result of damage to the host's own tissues
signalling danger to the immune system (Matzinger 1998; Medzhitov &
Janeway 2002). Pattern recognition is the first step in innate immunity.
Immunostimulants, like the presence of infection, are sensed by the various
PRRs. So far, 11 types of pattern recognition receptors have been identified in
shrimp namely, -1,3-glucanase-related proteins, -1,3-glucan-binding
proteins, c-type lectins, scavenger receptors, galectins, fibrinogen-related
proteins, thioester-containing Down syndrome cell adhesion molecules, serine
protease homologs, trans-activation response RNA-binding protein, and Toll-
like receptors. Aside from pattern recognition, these PRRs have different
binding specificities and effector functions (Wang & Wang 2013).
Immunostimulation can strengthen the immune system of farmed aquatic
animals and increase their resistance to pathogens during exposure to stress,
48 Mary Jane S. Apines-Amar & Edgar C. Amar

such as handling, crowding, sampling, transport, vaccination, during

reproduction, and also during the larval stages when high levels of mortality
occur. Some commercially available immunostimulants are shown in Table 1.

Sources of immunostimulants
Bacterial preparations
Vibrio - is a curved rod-shaped gram-negative bacteria. Vibrio
anguillarum is a very efficient vaccine for salmonid fish (Johnson et al 1982;
Norqvist et al 1989; Sakai et al 1995). The immunostimulant effects of
inactivated Vibrio cells have been documented in shrimp (Itami et al 1989,
1991, 1992; Horne et al 1995; Teunissen et al 1998; Pereira et al 2009; Powell
et al 2011; Lin et al 2013). The authors reported that injection or immersion of
shrimp in Vibrio bacterin resulted in reduced mortality suggesting
immunostimulation by the ”vaccine” as invertebrates do not have an efficient
specific immune response. The immunostimulation is mediated by the PRRs
that recognize and bind stimulatory components in bacteria. Vibrio bacterin
may therefore act as an immunostimulant since non-specific immune cells
such as phagocytic hemocytes are activated (Sakai 1999). A Vibrio harveyi
bacterin was also able to protect P. monodon against WSSV infection
(George et al 2006).
Lipopolysaccharide (LPS) - also known as lipoglycans or endotoxins
are large molecules consisting of a lipid and a polysaccharide joined by a
covalent bond. They are the major component of the outer membrane of
gram-negative bacteria, are responsible for the structural integrity of the
bacteria, and stimulate strong immune responses in recipient hosts. The
lipid component in association with the main polysaccharide is responsible
for the biological activities of LPS (Rietschel et al 1993). The
immunostimulant effects of LPS have been demonstrated in fish (Salati
et al 1987; Neumann et al 1995; Nya et al 2010) and shrimp (Vargas-
Albores et al 1998; Newman 2000; Felix 2005). LPS, at low doses,
improves disease resistance and acts as a prophylactic agent (Noworthy
1983). Rungrassame et al., (2013) found that shrimp fed LPS-containing
diet exhibited significantly higher survival rates when exposed to
V. harveyi than those fed the normal diet. Some crucial immune-related
transcripts such as anti-lipopolysaccharide factor 3 (ALF3), C-lectin and mucin-
like peritrophin were also induced in shrimp digestive tracts by the LPS
supplement. These findings led the authors to conclude that LPS
supplement is a promising candidate to increase disease resistance in black
tiger shrimp farming.
Use of immunostimulants in shrimp culture: An update 49

Live bacteria: probiotics as immunostimulants – A number of studies

revealed that the supplementation of probiotic bacteria and commercial
probiotics in feed or any sort of inclusion can boost the cellular and
humoral components of the innate immune system in several species of
fish and shellfish including salmonids and shrimps (Gullian et al 2004;
Panigrahi et al 2004, 2005, 2007; Song et al 2006; Balcazar et al 2006,
2007a, 2007b; Rodrıguez et al 2007; Pais et al 2008; Goncalves et al 2011;
Biswas et al 2013; Cerezuela et al 2013; Meena et al 2013; Perez-Sanchez
et al 2013; De et al 2014). Immunostimulation by Bacillus S11 bacteria
increased phagocytic activity in Penaeus monodon (Rengpipat et al 2000),
whereas the administration of Lactobacillus plantarum stimulated
phenoloxidase and superoxide dismutase activities leading to enhanced
clearance efficiency of Vibrio alginolyticus in Litopenaeus vannamei (Chiu
et al 2007).

Complex carbohydrates

Glucans - are natural biomolecules with immunomodulatory activity. In

vertebrates, glucans modulate the immune response through the macrophage
and dendritic immune cells. The amount or dose of glucans does not
determine its effectiveness as sources, processing, size and uniformity of
glucan particles are the actual determinants of its efficacy
(www.betaglucan.com). Enhancement of immune responses and protection
against infectious agents by treatment with beta-glucans were observed in
fish (Robertsen et al 1990; Raa et al 1992; Engstad & Robertsen 1993;
Sahoo & Mukherjee 2002; Selvaraj et al 2005; Rodriguez et al 2009; Skov
et al 2012; Meena et al 2013; Vetvicka et al 2013) as well as in shrimp (Song
& Hsieh 1994; Sung et al 1994; Chang et al 2000, 2003; Wang et al 2008).
In shrimp aquaculture, the most widely used immunostimulants are glucan-
based (Nahavandi et al 2010).
Prebiotics as immunostimulants – prebiotics are indigestible fibers that
increase beneficial gut commensal bacteria resulting in improvements of the
host’s health (Song et al 2014). Prebiotics such as fructooligosaccharide
(FOS), mannan oligosaccharide (MOS), inulin, or -glucan are called
immunosaccharides bacause they directly enhance innate immune responses
including phagocytic activation, neutrophil activation, stimulation of the
alternative complement system, and increased lysozyme activity (Song et al
2014).
50 Mary Jane S. Apines-Amar & Edgar C. Amar

Nutritional factors
Vitamins - dietary supplementation of vitamins such as A, C, and E is
effective in increasing the immunocompetence and disease resistance of
fish (Waagbo et al 1992; Mulero et al 1998; Verlhac et al 1998; Ortuno
et al 1999, 2000; Cuesta et al 2001; Sahoo & Mukherjee 2002; Tewary &
Patra 2008). Vitamins A, C, and E in combination with dietary carotenoids
were found to enhance the complement, lysozyme, and phagocytic
activities in fish (Amar et al 2001). Improved immune function and
resistance to pathogens were also demonstrated in several species of
penaeid shrimp that received vitamins A, C, and E supplements (Merchie
et al 1998; Lee & Shiau 2002, 2003, 2004; Nahavandi et al 2010;
Sivakumar & Felix 2011).
Carotenoids - the role of carotenoids in the immune response of
animals has been established (Chew 1993). Carotenoids improve
lymphocyte blastogenesis, lymphocyte cytotoxicity activity and stimulate
the production of certain cytokines. Carotenoids also stimulate the
phagocytic and bacterial killing ability of neutrophils and macrophages. In
fish, dietary carotenoids have been reported to heighten immune responses
and increase disease resistance (Christiansen et al 1995; Tachibana et al
1997; Amar et al 2000, 2001, 2004, 2012). Similarly in shrimp,
supplementation of dietary carotenoids resulted in increased resistance to
stress, salinity shock, and increased the antioxidant response before and
after viral infection (Merchie et al 1998; Nahavandi et al 2010; Pacheco
et al 2011).
Trace elements - deficiencies in trace elements with antioxidant
function (cofactors of numerous enzymes) such as zinc (Zn), copper (Cu),
manganese (Mn) and selenium (Se) have been reported to depress
immunity in animals. Deficiency in Zn, Mn and Cu were found to reduce
the natural killer-like activity of leukocytes and antibody production in fish
(Kiron et al 1993; Paripatananont and Lovell 1995a; Inoue et al 1998).
Dietary Zn and Se protected fish against Edwardsiella ictaluri infection
(Paripatananont and Lovell 1995b; Wang et al 1997). Enhanced lysozyme
activity and total immunoglobulin level, and improved antibody production
and survival were exhibited by rainbow trout supplemented with amino
acid-chelated trace elements (Apines-Amar et al 2004). Likewise,
supplementation of Cu in tiger shrimp resulted in improved growth, total
hemocyte count, and superoxide anion production (Lee & Shiau 2002). A
similar improvement of immune responses was observed in white shrimp
upon dietary suplementation with Zn (Lin et al 2013).
Use of immunostimulants in shrimp culture: An update 51

Animal sources/extracts
Chitin/Chitosan - chitin is one of the most abundant polysaccharides in
nature, and a common constituent of insects, crustacean exoskeletons, and
fungal cell walls (Esteban et al 2000). Chitosan is the product of alkaline
deacetylation of chitin obtained from crustacean shells. Injection, immersion,
and oral administration of chitin and chitosan have been reported to protect
fish and shrimp against bacterial infection (Sakai et al 1992; Siwicki et al
1994; Anderson et al 1995; Wang and Chen 2005; Huxley et al 2010, 2011).

Plant sources/extracts
Seaweeds - plants including seaweeds are rich sources of safe and
inexpensive chemical compounds. Products derived from plants exhibit
various activities and are utilized as anti-stress agents, growth promoters,
appetizers, tonics, immunostimulants and antimicrobials (Citarasu et al
2002; Immanuel et al 2004). Red seaweeds contain biologically active
sulphated, galactose-based polysaccharides, and show antibacterial and
antiviral activities (Renn 1997; Rajasulochana et al 2009; Wongprasert et al
2013). Rudtanatip et al (2014) reported that sulfated galactans from
Gracilaria fisheri elicits anti-WSSV activity by binding to viral proteins
which are essential structures for viral attachment to host cells. Fucoidan, a
sulfated polysaccharide from brown algae (e.g, Fucus, Laminaria) activated
the immune system and increased the resistance to viral infection in penaeid
shrimp (Nahavandi et al 2010). An extract from Gracilaria tenuistipitata
exhibited protection in shrimp against WSSV as evidenced by the higher
survival rate and heightened immune responses (Lin et al 2011). Likewise,
white shrimp Litopenaeus vannamei immersed in seawater containing brown
seaweed Sargassum hemiphyllum var. chinense powder or its extract showed
improved immunity and resistance against Vibrio alginolyticus and WSSV
infections (Huynh et al 2011).
Herbs – the benefits of herbs such as onion, ginger, and garlic on human
health are well-recognized. Enhanced immune responses and resistance to
diseases were recently demonstrated in fish and shrimp supplemented with
either onion, ginger, or garlic (Dugenci et al 2003; Nya & Austin 2009,
2011; Chang et al 2012; Apines-Amar et al 2012, 2013; Aruvasu et al 2013;
Haghighi & Rohani 2013; Talpur et al 2013; Kanani et al 2014). Elevated
expression of the immune-related genes Penaeidin, Crustin, Lysozyme,
Toll-receptor, and tumour necrosis factor was detected in kuruma shrimp
upon stimulation with garlic (allicin) extract (Tanekhy and Fall 2015). The
methanolic extracts of different herbal medicinal plants like Cyanodon
52 Mary Jane S. Apines-Amar & Edgar C. Amar

dactylon, Aegle marmelos, Tinospora cordifolia, Picrorhiza kurooa and

Eclipta alba were found to be effective against white spot syndrome virus
(WSSV) infection in shrimp (Citarasu et al 2006).

Hormones/cytokines
Growth hormone (GH) – is a peptide hormone (also known as
somatotropin or somatropin) that stimulates growth and cell reproduction in
humans and other animals. Growth hormone has numerous beneficial effects
such as promotion of lipolysis, gluconeogenesis, increase in protein
synthesis and stimulation of immune responses among others. The influence
of GH on the immune responses of fish have been studied (Harris & Bird
2000; Yada et al 2002, 2004, 2007; Peterson et al 2007). A recombinant
bovine growth hormone was found to enhance growth and immunity in
shrimp larvae (Kang et al 2005).
Lactoferrin (LF) - formerly known as lactotransferrin (LTF), is a
glycoprotein and a component of the innate immune system. Lactoferrin
influences the growth and proliferation of various pathogens such as
bacteria, viruses, protozoa, or fungi (Kirkpatrick et al 1971). LF affects
mainly the respiratory burst and natural cytotoxic activities in fish (Esteban
et al 2005).The antimicrobial activities of lactoferrin have been reviewed by
Adlerova et al (2008). In crustaceans, dietary administration of bovine
lactoferrin enhanced immune indices and resistance against Aeromonas
haydrophila challenge in Macrobrachium rosenbergii (Chand et al 2006).

Synthetic sources
Levamisole - known under the tradename Ergamisol, was originally
synthesized as an anti-helminthic and has been widely used as an
immunomodulator belonging to imidazothiazole derivatives. Levamisole has
been used in humans to treat parasitic worm infections, and has been studied in
combination with other forms of chemotherapy for colon cancer, melanoma,
and head and neck cancer. Oral administration of levamisole resulted in
enhanced immunity and improved resistance to diseases in fish (Siwicki 1987,
1989; Siwicki et al 1990; Kajita et al 1990; Sivakumar & Felix 2011). Oral
administration of levamisole likewise resulted in increased protection against
bacterial infection in tiger shrimp (Huxley et al 2010, 2011).
CpG Oligodeoxynucleotide (CpG ODN) – are short single-stranded
synthetic DNA molecules containing cytosine triphosphate deoxynucleotide
Use of immunostimulants in shrimp culture: An update 53

"C" followed by a guanine triphosphate deoxynucleotide "G" with

phosphodiester "p" linking the two nucleotides. CpG motifs are considered
pathogen-associated molecular patterns (PAMPs). CpG motifs are more
prevalent in bacterial DNA, and when unmethylated, function as
immunostimulants (Krieg et al 1995; Weiner et al 1997). In contrast, the DNA
of vertebrates are deficient in CpG motifs and highly methylated. Recently,
nucleotides have received much attention as potential immunomudulators. Li
& Gatlin (2006) has reviewed nucleotide nutrition in fish. The beneficial
influences of oral administration of nucleotides on immune functions, vaccine
efficiency or disease resistance have been demonstrated in fish (Ramadan et al
1994; Burrells et al 2001; Sakai et al 2001; Li & Gatlin 2006). It must be
pointed out that the nucleotides in the diets of farmed aquatic animals are
added to provide the supply of nucleotides (as nutrient) necessary to enhance
growth and survival especially under conditions of high requirement or
stressful events, whereas CpG is a PAMP and is a non-nutrient nucleotide.
Nevertheless, even nucleotides without CpG motifs can be sensed as non-self
molecules, such as in cases of autoimmune reactions. Increased immune
indices have been reported in shrimp injected with ODNs with or without CpG
motifs (Amar & Faisan 2012).

Table 1. Some currently available commercial immunostimulants.

54 Mary Jane S. Apines-Amar & Edgar C. Amar

Effects of immunostimulants

Upregulated immune indices such as total hemocyte count, respiratory

burst, phenoloxidase activity, phagocytic activity, agglutination titer, lysozyme
and SOD activities, etc. have been reported in many immunostimulation
studies (Table 2). Growth-promoting activity was also found with some
immunostimulants (Song & Hsieh 1994). Growth enhancement could result
from improved disease resistance due to immunostimulant supplementation.
Shrimp fed with peptidoglycan-supplemented feed also showed better growth
and feed conversion rates than those fed a normal diet (Boonyaratpalin et al.,
1995). Sung et al (1994) demonstrated that black tiger shrimp grew faster with
glucan immersion which could be attributed to the higher activity of glucan
delivered by immersion compared to oral administration.

Table 2. Various types of immunostimulants studied in shrimp.

Use of immunostimulants in shrimp culture: An update 55

Table 2. Continued
56 Mary Jane S. Apines-Amar & Edgar C. Amar

Table 2. Continued
Use of immunostimulants in shrimp culture: An update 57

Table 2. Continued

Methods and strategies in using immunostimulants

Immunostimulants can be administered through injection, immersion, or
oral administration. Injection and immersion require handling of fish/shrimp
or confining them in a small area during application. However, these
methods are laborious, time-consuming, and stressful. Injection method is
the most cost-effective method for large fish (>10g). Immersion on the other
hand, is not as effective as injection but allows mass immunostimulation and
is the most cost-effective method for smaller fish (<5g). Oral
immunostimulation is a non-stressful method that can be used with any size
of fish but requires a large amount of immunostimulant to provide
protection. Furthermore, this method is applicable only for fish fed artificial
diet.
58 Mary Jane S. Apines-Amar & Edgar C. Amar

The outcome of using an immunostimulant is usually determined by the

strategy by which it is applied (Bricknell & Dalmo 2005). Continuous use of
an immunostimulant may up-regulate the immune system and maintain this
status until the immunostimulant is withdrawn, or it may cause adverse
effects such as tolerance or immunosuppression. On the other hand, pulse
administration (administering immunostimulant-supplemented and
non-supplemented diets alternately) oscillate the immune response from a
resting level to a heightened response then back to resting, and has been
shown to be a better strategy of immunostimulant application.

Some issues in the use of immunostimulants in shrimp

Lack of correlation of in vitro and in vivo studies. Some
immunostimulants may enhance the non specific immune response in vitro
but this does not always result in improved health or increased survival.
Some immunostimulants do not show a linear dose/effect relationship; they
could be effective at a certain optimum concentration but have no effect or
exhibit toxicity at higher concentrations. Consequently, doses determined
in vitro cannot be directly extrapolated to large-scale production systems.
Hence, these immunostimulants may end up being fed at high doses or for
long durations resulting in chronic overstimulation and exhaustion of the
immune system.
There is lack of unequivocal evidence on the effficacy of some products.
Most studies on immune stimulation by microbial polysaccharides claim
beneficial effects by improving growth and resistance to pathogen challenge,
and/or stimulation of immune responses such as prophenoloxidase,
antibacterial, antioxidant, and agglutinaton activity, and reactive oxygen
production. However, these effects are short term, and there are very few
studies conducted in large-scale production units on a longer duration. With
particular immunostimulants, some studies reported beneficial effects,
whereas no positive effects were found by others.
Some immune responses are detrimental. Essentially, the haemocytes
perform inflammatory-type reactions such as phagocytosis, haemocyte
clumping, production of reactive oxygen metabolites, and the release of
microbicidal proteins. Under normal conditions, the inactive form of effector
molecules are stored in the hemocytes but are released into the hemolymph
through exocytosis and activated upon stimulation by non-self molecules. In
the open circulatory system of crustaceans, immune reactions must be
localized to avoid self-damage. Some of the responses and reactions that are
potentially self-harming include degranulation of hemocytes, cell clumping
and hemacytopenia, cytotoxicity at higher doses, depletion of immune
Use of immunostimulants in shrimp culture: An update 59

effectors, immune exhaustion by repeated immunostimulation, and high

energetic cost of regenerating immune components (Smith 2008). With these
possible adverse consequences, a rigorous product testing on a large scale
over a duration spanning the whole crop cycle is essential. The capacity of
the products to upregulate immunity genes should also be considered with
the objective of correlating in vitro and in vivo results. Lastly, stimulation
that elicits alternative molecules that have no self-harming potential but have
direct effect on the pathogens (such as antimicrobial peptides) should be
pursued.

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