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Research Signpost
37/661 (2), Fort P.O. Trivandrum-695 023
Kerala, India
Biotechnological Advances in Shrimp Health Management in the Philippines, 2015: 89-114
ISBN: 978-81-308-0558-0 Editors: Christopher Marlowe A. Caipang, Mary Beth I. Bacano-Maningas and Fernand F. Fagutao
5. Mechanisms of probiotic actions in
shrimp: Implications to tropical aquaculture
Carlo C. Lazado1, Jermaine I. Lacsamana2 and Christopher M.A. Caipang3 1Section for Aquaculture, National Institute of Aquatic Resources, Technical University of Denmark North Sea Science Park, 9850 Hirtshals, Denmark; 2Fisheries Policy and Economics Division, Bureau
of Fisheries and Aquatic Resources, 1101 Quezon City, Philippines; 3School of Applied Science, Temasek Polytechnic, 529757, Singapore
Abstract. The shrimp aquaculture industry had been very
dependent to synthetic antimicrobials as disease control agents. This approach was considered unsustainable and not eco-friendly because drug-resistance and horizontal transmission following excessive and indiscriminate use posed serious threats. The industry was then confronted to search suitable and effective alternatives, and the use of beneficial microorganisms or probiotics was identified to be very promising. Probiotics are live or dead, or even a component of microorganisms capable of
conferring beneficial effects to the host and/or its environment. Interestingly, probiotic actions are multifaceted in nature as benefits are delivered in several mechanisms. They are not only capable of protecting the farmed animals from diseases but they can also influence growth, nutritional status and the quality of the rearing environment. The application of probiotics in shrimp has been demonstrated to 1) influence the soil and water quality, 2) enhance growth, 3) inhibit pathogen proliferation, 4) modulate the
Correspondence/Reprint request: Dr. Carlo C. Lazado, Section for Aquaculture, National Institute of Aquatic
Resources, Technical University of Denmark, North Sea Science Park, Hirtshals, Denmark
E-mail: [email protected]
Carlo C. Lazado et al. 90
host microbiota, 5) contribute essential nutrients and beneficial enzymes, and 6) modulate the host immunity. The chapter synthesizes the identified mechanisms of probiotic actions in shrimp and provides future perspectives in support of the continual search and development of probiotics for shrimp aquaculture. In addition, a microcosm discussion is provided citing the current status and issues on probiotics research and applications in Philippine shrimp aquaculture. This paper affirms the importance of probiotics in the effective health management of shrimp aquaculture.
Introduction
Shrimp aquaculture is a profitable venture but its economic potential is
challenged by several issues and concerns that considerably hinder
sustainable growth and development. Viral and bacterial diseases are among
the commonly associated limiting factors of a successful shrimp aquaculture.
The use of antibiotics was a conventional strategy to prevent and control
disease outbreaks. There had been an excessive and indiscriminate use of
antibiotics (Mohapatra et al., 2013), and this practice still persists until now
despite several precautionary standards set in a number of countries and
politico-geographical regions. One of the biggest concerns was the development of drug-resistance and multiple antibiotic resistance (MAR) in
bacteria, which resulted in resistance transfer and reduced efficacy of
antibiotic treatment (Tendencia and de la Peña, 2001). In addition, the
prevalence of antibiotic residues in the ponds was considered a serious
environmental risk. Following incidences primarily on these concerns, the
excessive use of antibiotics as disease control agents in shrimp aquaculture
had been reduced and efforts in searching alternative strategies were
encouraged.
The exploration of alternative strategies paved way to the development
and application of probiotics as a thematic aspect of health and welfare
management in aquaculture. In light of new trends towards eco-friendly
aquaculture production, the promise of probiotics was welcomed with positivity as evidenced by numerous papers discussing and advancing their
beneficial properties, relevance and application (Guo et al., 2009; Lazado &
Caipang, 2014a; Ninawe & Selvin, 2009; van Hai & Fotedar, 2010).
Probiotics was primarily considered a disease control agent in shrimp
aquaculture. In this disease control context, probiotics have been
demonstrated capable of directly inhibiting the growth and proliferation of
pathogens and also stimulating the dominance of the beneficial microbiota of
the host and its rearing environment (Chythanya & Karunasagar, 2002;
Lazado & Caipang, 2014c; Lazado et al., 2011; Lazado et al., 2010b; Li et al.,
2007; Preetha et al., 2007; Vaseeharan and Ramasamy, 2003). It is important
Mechanisms of probiotic actions in shrimp: Implications to tropical aquaculture 91
to mention that the host animal and its immediate environment impose
decisive factors for these benefits to be delivered optimally. Thus, the selection of appropriate probiotics particularly by taking into consideration
the physiological and biochemical features of the host and the physico-
chemical conditions of the immediate environment is crucial (Lazado et al.
2015). Interestingly, several other beneficial properties have been
documented in probiotics hence expanding the understanding of their use in
farmed aquatic animals. The application of probiotics has a significant
advantage not only by being a disease control agent but notably as
alternatives that promote health and welfare in farmed animals in a larger
scope (Lazado & Caipang, 2014a).
The use of microorganisms and their metabolites is not a new approach
in health management. Nevertheless, one of the major rationales affirming
the importance of probiotics in aquaculture is its credibility in fostering sustainability by having insignificant risk of harm to the farmed animals and
their environment. Most importantly, they offer effective and viable
alternative solution to a problem impeding the industry. The chapter
provides a synthesis of the current knowledge on the applications of
probiotics in shrimp focusing on how this group of microorganisms confers
their benefits to the host and its environment. Several probiotic mechanisms
have been identified through the years, but this chapter discusses how they
1) improve the soil and water quality, 2) enhance growth, 3) inhibit
pathogens, 4) influence host microbiota, 5) contribute nutrients and
enzymes, and 6) modulate host immunity. At the end of the chapter, a
separate discussion is provided on the current status of probiotics research and application in the Philippines.
Probiotics as beneficial microorganisms. Probiotics is coined from the
Latin word PRO meaning for and from the Greek word BIOS meaning life
(Zivkovic, 1999). The word probiotics was introduced by Lilly and Stillwell
in 1965 as substances produced by a microorganism that prolong the
logarithmic growth phase in other species (Lilly & Stillwell, 1965). The idea
was re-introduced in the context of microbial feed/food supplements by
Parker in 1974 (Parker, 1974). One of the most significant definitions of
probiotics was coined by Fuller by defining probiotics as “live microbial
food supplement that benefits the host (human or animal) by improving the
microbial balance of the body” (Fuller, 1989). This definition of probiotics
had been the most popular and widely refered to both in human and veterinary studies for several years (Lazado & Caipang, 2014a).
The empirical concept of probiotic application in aquaculture was made
by Kozasa in mid 1980s when he used the spores of Bacillus toyoi as feed
additive to increase the growth rate of yellow tail, Seriola quinqueradiata
Carlo C. Lazado et al. 92
(Kozasa, 1986). However, it did not trigger much attention as evidenced by
the insignificant amount of research studies published within that aspect thereafter. Probiotics research started to become very prominent in
aquaculture during the late 1990s. This approach was evoked when the
active call for alternatives to antibiotics had become very resolute. In the
early 2000s, Verschuere and his colleagues proposed an aquaculture-based
definition stating that “A probiotic is defined as a live microbial adjunct
which has a beneficial effect on the host by modifying the host-associated or
ambient microbial community, by ensuring improved use of the feed or
enhancing its nutritional value, by enhancing the host response towards
disease, or by improving the quality of its ambient environment”
(Verschuere et al., 2000). This definition allowed a broader application of
the term “probiotic” and addressed concerns on Fuller’s definition
particularly on the complex interaction between the culture environment and the host that characterized the aquatic environment. A joint expert panel
from Food and Agriculture Organization of the United Nations/World Health
Organization (FAO/WHO), defined probiotics as “live microorganisms,
which when consumed in adequate amounts, confer a health benefit for the
host” (FAO/WHO, 2001). The complexity of the aquatic environment makes
these pioneering definitions disputable and their application could be on a
case-by-case basis (Lazado & Caipang, 2014a). This is particularly relevant
if we introduce the concepts of 1) application to the rearing water (Makridis
et al., 2005; Spanggaard et al., 2001) and 2) bacterial viability (Díaz-Rosales
et al., 2006; Lazado & Caipang, 2014b; Lazado et al., 2010a; Salinas et al.,
2006). The unprecedented amount of researches generated on probiotics in aquaculture put forth the need to have a working definition in an aquaculture
point of view. Necessary considerations must be made in order that a
definition encompasses the physico-chemical and biological prerequisites of
a dynamic aquatic system including the organisms thriving in it. In response
to the point raised by Merrifield et al. (2010) that abovementioned concerns
need to be considered in the understanding of probiotics for farmed aquatic
animals, a simplified yet broad definition of probiotics was proposed by
Lazado & Caipang (2014a) stating that “live or dead, or even a component
of the microorganims that act under different modes of action in confering
beneficial effects to the host or its environment”. This definition could be
applied both in fish and in penaeids. For this chapter, the term “probiotics” is
referenced to the aforementioned definition. Mechanisms of probiotic actions. Interestingly, probiotics application
is not an approach based on a single mechanism. In fact, these beneficial
microorganisms are capable of delivering their benefits in scores of means
(Farzanfar, 2006; Gomez-Gil et al., 2000; Lazado & Caipang, 2014a,d;
Mechanisms of probiotic actions in shrimp: Implications to tropical aquaculture 93
Mohapatra et al., 2013; Ninawe & Selvin, 2009). The probiotic action can
either be direct (i.e. host) or indirect (i.e. environment) or a combination of both. This broad perspective of probiotic action is a product of a diversified
understanding of probiotics in an aquatic setting. It is now withdrawing from
the traditional understanding of being just disease control agents. The
principal consideration that probiotics as disease control agents will still
persist however their other mechanisms of action provide cognizance of their
numerous applications. The beneficial effects of probiotics are products of
several interrelated or dependent mechanisms.
Most studies on probiotic applications in aquaculture were conducted in
fish (Lazado et al. 2015). Nevertheless, there are still considerable amount of
researches in shrimp demonstrating the mechanisms observed in the piscine
model. In the succeeding sections, documented mechanisms of probiotic
actions in penaeid model are enumerated and described. A pictorial representation of the different mechanisms highlighting the interrelationship
of the probiotic actions is given in Figure 1.
Improvement of soil and water quality. The physico-chemical
properties of the rearing soil and water are crucial for the success of shrimp
culture. For instance, persistent infections could be actually due to poor
water quality and low water exchange rates (Zokaeifar et al., 2014). The
significance of environmental conditions can be introduced in the discussion
of probiotics in two ways: 1) optimum conditions support the biological
requirements of the host making them less susceptible to diseases, and 2) the
quality of the rearing system should promote the dominance of beneficial
microorganisms rather than the pathogenic population. One of the indirect mechanisms of probiotic actions in shrimp is the improvement of the culture
pond conditions especially modifying the physico-chemical properties of
water and soil.
The genus Bacillus is one of the widely known shrimp probiotics
capable of modifying the quality of the rearing environment. Bacillus spp.
are generally more efficient in converting organic matter back to CO2 than
other gram-negative bacteria, which would convert a greater percentage of
organic carbon to bacterial biomass or slime (Stanier et al., 1963). Bacillus
spp. were oftentimes used when the main aim of probiotic application was to
manipulate the rearing conditions. Bacillus were administered either
freeze-dried or microencapsulated to the rearing water of shrimp larval and
post-larval stages and were shown to decrease the level of ammonia, nitrite and pH of the water (Nimrat et al., 2012). There was also a reduction of
water ammonia, nitrite, nitrate and phosphate in the water of Penaeus
monodon culture pond treated with a commercial probiotics (Lakshmanan &
Soundarapandian, 2008). The reduction of ammonia and nitrite is attributed
Carlo C. Lazado et al. 94
to the capacity of the members of the Bacillus genera in mineralizing
nitrogenous wastes via nitrification and/or dinitrification (Joong et al., 2005; Zhao et al., 2010). Further, the nitrification process discharges hydrogen ion
leading to a reduction in pH (Camargo & Alonso, 2006). Probiotic addition
can also modify the chemistry of benthic community as shown by the
lowering of total nitrogen and total carbon concentrations in P. monodon
culture ponds (Shishehchian et al., 2001). Application of commercial
probiotics composed of Bacillus sp., Nitrosomonas sp., Nitribacter sp. and
Lactobacillus to Penaeus vannamei ponds resulted to the mitigation of
nitrogen and phosphate pollution in pond sediments as shown by the
reduction of total phosphorus, total inorganic phosphorus, total nitrogen and
total organic carbon (Wang & He, 2009).
Figure 1. Mechanisms of probiotic actions in shrimp. Probiotics are known to confer
benefits through different mechanisms. These beneficial microorganisms are capable of producing inhibitory substances that could interact directly to bacterial and viral pathogens (Iyapparaj et al., 2013; Nhan et al., 2010; Vaseeharan and Ramasamy, 2003). Probiotics are also capable of manipulating the pond ecosystem such as the microflora (Boonthai et al., 2011) and the physico-chemical conditions (Zokaeifar et al., 2014). Dietary supplementation of probiotics enhances growth (Johansson et al., 2000) through enzyme contribution that increases digestibility (Leonel Ochoa-Solano and Olmos-Soto, 2006). Probiotics are also capable of manipulating the host
microbiota (Makridis et al., 2005; Preetha et al., 2007). The protection from pathogens after probiotic treatment could be attributed to the direct inhibition of pathogens or to the capability of probiotics in modulating the immune system of shrimp (Chiu et al., 2007; Fu et al., 2011).
Mechanisms of probiotic actions in shrimp: Implications to tropical aquaculture 95
Pathogen inhibition. The direct inhibition of pathogens via probiotics
could be through the production of antagonistic metabolites (Chythanya and Karunasagar, 2002; Iyapparaj et al., 2013; Lazado et al., 2010b) and/or
interference of adhesion (Lazado et al., 2011). These characteristics are
commonly used as primary tests in selecting and identifying candidate
probiotics. Most of the probiotics in shrimp are characterized by their
capability to inhibit the growth of a wide range of pathogens. An isolate
identified as Pseudomonas sp. from the digestive tract of healthy juvenile
Litopenaeus vannamei exhibited strong antibacterial activity against Vibrio
parahaemolyticus, V. alginolyticus and V. cholera (Far et al., 2013). A novel
strain of Bacillus pumilus was isolated from the midgut of healthy black
tiger shrimp and showed strong inhibitory activity against V. alginolyticus,
V. mimicus, and V. harveyi. In addition, the suitability of the isolate as
probiotics was supported by the absence of any known B. cereus enterotoxin genes (Hill et al., 2009). Several candidate Bacillus probiotics demonstrated
strong inhibition on the growth of pathogenic V. harveyi strains isolated
from different origins (Thailand, Philippines and LMG4044). It was further
shown that toxin production of V. harveyi is decreased by the addition of
B. licheniformis or B. megaterium supernatant (Nakayama et al., 2009).
One of the areas in the study of probiotics in shrimp that needs to be
explored further is the identification of the antagonistic metabolites. Works
on the identification of antagonistic activity of candidate probiotics in most
cases halted after observing the inhibitory activity, thus often failed to
explore the identity of the metabolites that render such effect. Pseudomonas
I-2 strain exhibited strong inhibitory activity against several strains of pathogenic Vibrios. The inhibitory compound produced was characterized as
low molecular weight, heat stable, soluble in chloroform and resistant to
proteolytic enzymes (Chythanya & Karunasagar, 2002). Lactobacillus sp.
MSU3IR, a goat milk isolate and a candidate shrimp probiotics, produced
bacteriocin as an inhibitory substance. The production of bacteriocin of this
isolate could be optimized by manipulating the culture conditions of the
bacteria (Iyapparaj et al., 2013).
Quorum sensing was demonstrated as one of the virulence mechanisms of
many pathogenic bacteria including V. harveyi (Lilley & Bassler, 2000). This
bacterial phenomomen allows single bacterial cells to measure the
concentration of bacterial signal molecules and works in either of the two
known systems: the Autoinducer 1 system (AI-1) for the intraspecies communication using different Acyl-homoserine lactones (AHL) or AI-2 for
the interspecies communication (Jacobi et al., 2012). Application of N-acyl
homoserine lactone-degrading bacterial enrichment cultures (EC) directly to
the water or via Artemia nauplii of Macrobrachium rosenbergii elicited
Carlo C. Lazado et al. 96
protective effect following V. harveyi infection. The quorum quenching
feature of the EC is attributed for this observed protection (Nhan et al., 2010). Microorganisms capable of producing compounds that inhibit the quorum-
sensing signals could be established as new generation of antimicrobial agents
that would be useful for veterinary science and agriculture (Chu et al., 2011).
In an in vivo setting, the antagonistic mechanism of probiotics was
demonstrated by the decrease in number of the presumptive pathogenic
group in the rearing environment. The luminous bacterial count in the
rearing system of P. monodon decreased significantly after probiotic
treatments (Janeo and Corre Jr, 2011). The presumptive count of Vibrio in
the rearing water decreased significantly in the shrimp treated with
probiotics with known antagonistic activity (Boonthai et al., 2011). In
another study, the colonization of V. harveyi in P. monodon was inhibited
significantly by exposure to probiotics and the mechanism of inhibition was by competitive exclusion (Gullian et al., 2004).
Most of the inhibition studies used bacteria as pathogen targets.
However, some reports showed that the inhibitory spectrum of probiotics is
not only limited to bacterial pathogens as they have demonstrated antiviral
properties as well. Pediococcus pentosaceus isolated from the gut of wild
brown shrimp (Farfantepeneus californiensis) were administered through
feeds to L. vannamei to determine its antiviral activity. Probiotic
administration showed a decrease in the prevalence of white spot syndrome
virus (WSSV) but not the hematopoietic necrosis virus (IHHNV) in naturally
infected shrimp (Leyva-Madrigal et al., 2011). Protective effect against
WSSV was also demonstrated in another study in L. vannamei using Bacillus OJ as probiotics. Increasing dosage of probiotics resulted in an
increasing trend in shrimp survival. Interestingly, a positive synergistic
effect was observed when probiotics were administered with 0.2%
isomatooligosaccharides (Li et al., 2009). Using a recombinant technology
approach, Bacillus subtilis spores harboring a viral protein VP28 was given
to L. vannamei and extremely high survival (83.3%) was observed following
challenge with WSSV (Fu et al., 2011).
Growth enhancement. The most popular, common and practical
method of probiotic application in aquaculture is through oral administration
as feed supplements (Gomes et al., 2009; Huang et al., 2006). Not
surprisingly, their growth-enhancing property was also explored. There is no
universal consensus on how probiotics influence growth of cultivated species including fish and shrimp. However, this beneficial influence is being
proposed to be an outcome of the increase in the appetite, or in the
improvement of digestibility (Martinez Cruz et al., 2012), wherein the latter
is the most explored in shrimp probiotic studies.
Mechanisms of probiotic actions in shrimp: Implications to tropical aquaculture 97
The widely used probiotics in shrimp with growth-enhancing capability
belongs to the Bacillus genera. It was shown that administration of Bacillus sp. in L. vannamei influenced the apparent digestibility coefficients (ADC)
of dry matter, crude protein, lipid, phosphorus, essential amino acids,
non-essential amino acids and fatty acids (Lin et al., 2004). Further, the
significant increase in the ADC was dependent on the concentration of the
probiotics in the administered diets. The influence of probiotics in the
growth performance of shrimp was also observed in Fenneropenaeus indicus
fed with commercial Bacillus probiotic cocktail (Ziaei-Nejad et al., 2006).
The feed conversion ratio and specific growth rate were significantly higher
in probiotic-fed group than the control group. In another study using Bacillus
isolates from the intestine of the host, morphometric changes were observed
in M. rosenbergii fed with probiotics. Besides the increase in weight, the
body length of probiotic-fed prawn was significantly longer than those in the control group (Deeseenthum et al., 2007). These significant changes were
also exhibited in a study with P. monodon (Soundarapandian & Sankar,
2008). Weight gain as an indicator of growth enhancement by probiotic
feeding was also demonstrated in P. monodon (Rengpipat et al., 1998;
Rengpipat et al., 2003), P. vannamei/L. vannamei (Gómez & Shen, 2008;
Gullian et al., 2004; Wang and Gu, 2010; Wang, 2007; Zokaeifar et al.,
2012), M. japonicus (Dong et al., 2013), M. rosenbergii (Mujeeb Rahiman
et al., 2010) and F. brasiliensis (De Souza et al., 2012). Probiotics could
facilitate the efficient utilization of nutrients and minerals from the feed
which eventually being used by the host for their biological requirements.
For example in Penaeus indicus fed with lactic acid bacteria, there was almost 20% increase on the digestibility when the shrimp were fed with
probiotics (Fernandez et al., 2011). Indeed, the observed changes in growth
performance could be attributed to the enhancement of different parameters
like feed efficiency and feed conversion efficiency (Boonthai et al., 2011;
Zokaeifar et al., 2014; Zokaeifar et al., 2012).
Enzymatic contributions. In relation to the capability of probiotics in
enhancing growth performance, their influence on the enzymatic physiology
of the gut is believed to be one of the significant contributors of this
beneficial outcome. The nutritional condition of farmed animals is a
consequence of both the given feeds and their digestive physiology (Becerra-
Dórame et al., 2012). Digestive enzymes are necessary to break complex
compounds into simpler and absorbable molecules that could be utilized by the host (Lazado et al., 2012). In fact, the utilization of the feed-related
substances is greatly dependent on whether they can be easily absorbed or
not by the host and used ultimately for their physiological processes. In some
cases, the digestive physiology of the host could be influenced by several
Carlo C. Lazado et al. 98
factors such as the environment, types of feed and health status. In the
context of probiotic applications, enzymatic influence could either be through stimulation (i.e. promotion of endogenous enzyme production) or
direct contribution (i.e. production of exogenous enzymes by the
administered microorganism).
Protein is considered a major limiting nutrient for shrimp growth (Kureshy
and Allen Davis, 2002), therefore probiotics producing proteases are regarded as
potential candidates. The increase in feed digestibility associated to probiotic
administration has been linked to the capability of these microorganisms in
facilitating the digestion of protein constituents through their enzymes that
contribute to the host endogenous proteolytic activity (Leonel Ochoa-Solano and
Olmos-Soto, 2006). Concomitant to the improvement in growth, significant
increase in digestive protease due to probiotic application was observed in
P. vannamei (Gómez & Shen, 2008; Liu et al., 2009; Wang, 2007) and Fenneropenaeus indicus (Ziaei-Nejad et al., 2006). Besides protease, probiotics
can also influence other digestive enzymes of shrimp such as amylase (Castex
et al., 2008; Gómez & Shen, 2008; Wang, 2007; Ziaei-Nejad et al., 2006),
cellulase (Wang, 2007), lipase (Wang, 2007; Ziaei-Nejad et al., 2006) and
trypsin (Nimrat et al., 2013). The influence of probiotics on the digestive
physiology of shrimp is continuously considered a desirable feature because their
contribution has associated positive consequences.
Modulation of host microbiota. The role of the host gastrointestinal
tract microbiota can be divided into 2 major distinctive functions. First is on
the context of immunity as the microbiota is crucial for the maintenance of
the mucosal immunity and serves as an important defensive barrier against invading pathogens (Lazado & Caipang, 2014a,d). The second function is on
nutrition as these commensal microorganisms provide nutrients and
beneficial enzymes to the host (Lazado et al. 2015; Nayak, 2010b).
Probiotics are known to modulate the microbial community structure of
the host. As discussed in a recent review paper in Atlantic cod, the discussion
of probiotics and gut microbiota should not be mutually exclusive. This is
because gut microbiota is a determinant of the success of probiotic
applications while probiotics are modulators of the gut microbial community
(Lazado & Caipang, 2014a). This inference is particularly interesting in
aquaculture species where application of probiotics is mainly through diets and
the gastrointestinal tract is the organ where host-probiotics interaction is highly
prominent. There are two central philosophies on how probiotics influence the microbial community structure of the host: 1) determinism, which states that a
well-defined dose-response relationship is required; and 2) stochasticism,
which describes chance favors organisms which happen to be in the right place
at the right time (Verschuere et al., 2000).
Mechanisms of probiotic actions in shrimp: Implications to tropical aquaculture 99
It was suggested by Dempsey that one or two phylogenetic groups
dominate the shrimp gut and have very low diversity (Dempsey et al., 1989). The profile shifts in the microbial structure following probiotic applications
can easily be observed because primarily, the normal gut microbiota of shrimp
is not as diverse as compared to fish. Shrimp fed with a commercial probiotic
cocktail (contained different strains of Bacillus) revealed the shift from the
dominance of Vibrio to Bacillus in the gut after 2 h and after 24 h in the
hepatopancreas (Janeo & Corre Jr, 2011). The change on microbial community
structure was also observed in L. vannamei exposed to probiotics of different
origins. The gut microbial community of the probiotic fed group was mainly
consisted of α- and γ-proteobacteria, fusobacteria, sphinobacteria and
flavobacteria while the control group was principally dominated by
α-proteobacteria and flavobacteria (Luis-Villaseñor et al., 2013). Though there
was no significant change in the total bacterial count in L. vannamei-fed with B. licheniformis, the application of probiotics lowered the Vibrio count in the
gut tract of shrimp (Li et al., 2007). This significant reduction of Vibrio in the
gut was also documented in P. monodon fed with Synechocystis MCCB
(Preetha et al., 2007) and in Marsupenaeus japonicus fed with Bacillus (Dong
et al., 2013). The administration of probiotics also increased the total microbial
count and decreased the population of Vibrio in the gut of P. japonicus (Zhang
et al., 2011). These observations exemplified the competitive exclusion
mechanism of probiotics in modifying the microbial population of the gut
specifically by lowering the count of the pathogenic group.
Immunomodulation. The interest on the importance of probiotics to
host immunity became very prominent in the early 1990s when significant evidence demonstrated several positive health-related responses to the
manipulation of the gastrointestinal microflora (Huis in 't Veld, 1991;
Smirnov et al., 1993). The studies in human models became the baseline
information in exploring the same effects in fish and other invertebrates
including shrimp. Particularly in fish, probiotic application can modulate
both the adaptive and innate immunity (Lazado & Caipang, 2014d; Nayak,
2010a). Shrimp are believed to be lacking an adaptive immunity but they
still possess an interesting innate immune system that effectively protects
them from harmful microorganisms (Young Lee & Söderhäll, 2002). Reports
showed that shrimp innate immunity responded to probiotic treatments
through the modulation of the cellular and humoral immune responses
(Lazado at el. 2015; Lakshmi et al., 2013; Ninawe & Selvin, 2009; van Hai & Fotedar, 2010). Though the current knowledge is fragmentary on how
probiotics stimulate the innate immunity of shrimp, a growing number of
researches provide, to some extent, the immunomodulatory mechanisms of
probiotics through treatment-response approaches. There are two apparent
Carlo C. Lazado et al. 100
reasons why the immunomodulatory features of probiotics have been of
great interest in shrimp aquaculture in the last years. During the early days of applications in shrimp, protection observed following pathogen challenge
was commonly used gauge of a positive consequence of probiotics
application. This result was traditionally explained through the direct
antagonism of the probiotics towards the pathogens (Figure 1). By looking
into a different perspective, this could be described by the capability of
probiotics in boosting the immunity to elicit potent responses against the
pathogens (Figure 1). Secondly, sustainable aquaculture is gearing towards a
more pro-active management. The immunomodulatory capabilities of
probiotics have been considered an effective prophylactic strategy. Fore
mostly, probiotics are greatly considered alternative to antibiotics and this
attribute made their application more enviable (Figure 2).
Figure 2. Model of immunomodulation in shrimp by probiotics. Pattern recognition
receptors are necessary in identifying microbe associated molecular patterns (PAMPs) of both the pathogens and the probiotics. Though several PAMPs have already been identified in shrimp, none of them have been demonstrated so far to be involved in the probiotics-host recognition in this species (Lazado et al., 2015). The application of probiotics influences the humoral and cellular defenses of the shrimp. Following probiotic treatment, increase in the number of hemocytes can be observed (Rengpipat et al., 2000; Zhang et al., 2011). Probiotics stimulate the production of reactive oxygen species (Mujeeb Rahiman et al., 2010). In addition, probiotic
treatment increases phenoloxidase activity (Chiu et al., 2007; Gullian et al., 2004) and hemocytic phagocytosis (Rengpipat et al., 2000; Tseng et al., 2009). The transcription of several immune-related genes can also be modulated by probiotic treatment (Antony et al., 2011; Dong et al., 2013). These stimulated immune defenses play a crucial role in the responses and eventual protection during pathogen exposure.
Mechanisms of probiotic actions in shrimp: Implications to tropical aquaculture 101
Hemocytes play important functions in shrimp immune responses
including recognition, phagocytosis, melanization, cytotoxicity and cell–cell communication (Johansson et al., 2000). They are characterized as hyaline
cell, small-granule cell and large-granule cell (Heng & Wang, 1998; Tsing
et al., 1989). Studies on the effects of probiotics in shrimp immunity are
mainly focused on the responses of hemocytes. Total hemocyte count (THC)
was increased following probiotic application in M. rosenbergii (Mujeeb
Rahiman et al., 2010), P. monodon (Rengpipat et al., 2000), P. japonicus
(Zhang et al., 2011) and L. vannamei (Li et al., 2007). However, it was
observed in L. vannamei that THC decreased significantly following feeding
with Lactobacillus plantarum (Chiu et al., 2007); no significant effect was
observed after feeding with B. subtilis (Tseng et al., 2009). This effect was
also observed in Pseudomonas-fed P. monodon (Alavandi et al., 2004).
Phagocytosis is a mechanism of most cells including hemocytes in combating and eliminating pathogens. In an interesting study, hemocytic
phagocytosis was enhanced by B. subtilis harboring a viral protein (VP28)
and this was speculated as a significant factor in the protection observed
against WSSV infection (Fu et al., 2011). Hemocytic phagocytosis was also
enhanced in P. monodon fed with Bacillus S1 (Rengpipat et al., 2000) and
L. vannamei fed with B. subtilis E20 (Tseng et al., 2009). In addition,
respiratory burst of hemocytes could also be enhanced by probiotic addition
(Mujeeb Rahiman et al., 2010).
Despite the absence of immunoglobulins, shrimp have developed
complex immune defenses that could counteract intruding pathogens and one
of them is through the prophenoloxidase (proPO) cascade system (Fagutao et al., 2009). Phenoloxidase plays a pivotal function in controlling bacterial
load in the haemolymph (Fagutao et al., 2009), in self non-recognition
(Hernández-López et al., 1996) and protection against pathogenic bacteria
(Amparyup et al., 2009). The application of probiotics resulted to the
stimulation of phenoloxidase activity in L. vannamei/P. vannamei (Chiu
et al., 2007; Li et al., 2007; Nimrat et al., 2013; Tseng et al., 2009; Wang &
Gu, 2010), P. monodon (Rengpipat et al., 2000), P. japonicus (Zhang et al.,
2011) and M. rosenbergii (Mujeeb Rahiman et al., 2010). In most cases, the
increase in the activity of phenoloxidase was concurrent to the protection
after pathogen challenge. Probiotics were also shown to modulate the
activity of other innate immune defenses such as superoxide dismutase
(Castex et al., 2009; Gullian et al., 2004; Li et al., 2007; Wang and Gu, 2010; Zhang et al., 2011), catalase (Castex et al., 2009), lysozyme and nitric
oxide synthase (Zhang et al., 2011). In addition, probiotic treatment did not
only enhance the antioxidant defenses of shrimp but it could also alleviate
Carlo C. Lazado et al. 102
oxidative stress (lower malondialdehyde and carbonyl protein) induced by
pathogen exposure (Castex et al., 2009, 2010). Immunomodulatory functions of probiotics were also demonstrated in
their capacity to influence the transcription of several immune-related genes
in shrimp. Upregulated expression was observed in prophenoloxidase
paralogs, antimicrobial peptides, peroxinectin, serine protease,
lipopolysaccharide-and β-1,3-glucan binding protein and lysozyme (Antony
et al., 2011; Dong et al., 2013; Liu et al., 2010; Zokaeifar et al., 2014;
Zokaeifar et al., 2012). The stimulated expression of these immune genes
was implicated to the observed robust responses and protection during
pathogen challenge and stress exposure.
Development of probiotics. The selection, evaluation and development
of probiotics is a multi-step procedure predominantly focused on their safety,
functionality and technological characteristics (Reid, 2006; Sanders & Huis In't Veld, 1999; Verschuere et al., 2000). Probiotics applications in
aquaculture had been for some time dependent on the use of terrestrial
probiotics especially the lactic acid bacteria (LAB). Though there is no
universal acceptance that probiotics should be from the host (Verschuere
et al., 2000), the utilization of host-derived microorganism has become a
popular and enviable approach at present (Lazado et al., 2015; Lazado &
Caipang, 2014a). There is no standard protocol that is universally accepted
and approved on the development of probiotics in shrimp though Lakshmi
and company proposed a procedure (Lakshmi et al., 2013) principally based
on previously published suggested strategies (Balcázar et al., 2006;
Verschuere et al., 2000). The approach presented is good however it did not show the importance of ensuring the identity of the microorganisms which
was suggested in the FAO-WHO guidelines of 2002 (FAO/WHO, 2002).
Secondly, it did not highlight the importance of both in vitro and in vivo
approaches in profiling the probiotic features of the candidate
microorganism. The multi-step approach especially in delineating the
in vitro and in vivo features is among the core areas of probiotic
development. Figure 3 shows a simplified proposed strategy for probiotic
development in shrimp, taking into consideration what have been done in
shrimp and some issues that should be addressed. The 2 main elements, both
mutually inclusive, on having a systematic approach in developing
probiotics for aquaculture are: 1) biological aspect: it is essential that the
candidate microorganism is identified properly and the mechanisms of probiotic actions are known; 2) commercial aspect: solid evidences on the
biological activities of the candidate microorganisms will substantiate the
claim and will serve as deciding factors to be granted authorization by the
regulatory agencies. In general, contemporary researches on the
Mechanisms of probiotic actions in shrimp: Implications to tropical aquaculture 103
development of probiotics for aquaculture only reached the biological aspect
and they did not advance into a commercial commodity as envisioned. In a global perspective, there are no international standard and
regulatory guidelines for the usage of probiotics in aquaculture (Ninawe &
Selvin, 2009). Usually, the regulatory agency of each country is the one
setting the guidelines on the use of probiotics in aquaculture. For example
in the European Union, the European Food Safety Authority set the
guidelines and Bactocell®, a lyophilized live Pediococcus acidalactici was
the first probiotics that was given approval for use in aquaculture
(E.F.S.A., 2012).
Probiotics in a Philippine perspective. In 2004, the Bureau of Food
and Drug Administration (now Food and Drug Administration) released
Bureau Circular No. 16 stating the guidelines of probiotic drug use in the
Philippines (BFAD, 2004). It was enumerated in the order that the approved bacterial strains as probiotics in the Philippines are the
following: Lactobacilli, Bifidobacteria, nonpathogenic strains of
Streptococcus, Saccharomyces boulardi and Bacillus causii. The order was
particularly directed for human application and did not mention its
applicability for veterinary use in the country. The main law governing
Philippine fisheries is Republic Act 8550 also known as The Philippine
Fisheries Code of 1998. The implementing rules and guidelines of this act
did not mention any provisions on the application of probiotics in
aquaculture. It is understandable that this law did not able to cover the
applications of beneficial microorganisms because at the time of drafting
until promulgation, probiotics was just an emerging thematic area in aquaculture. To our knowledge, there is no existing law or implementing
guidelines governing research and application of probiotics for aquaculture
species in the Philippines. This is one of the main challenges concerning
the status of probiotics in Philippine aquaculture. In reference to Figure 3,
one of the main components in the development of probiotics is its/their
compliance to laws or existing guidelines. If we follow this systematic
approach, it could not be achieved in the Philippines at present because
there are no rules that will guarantee this provision. Even though the
national veterinary drug residues control programme is under R.A. 3720 or
the “Food, Drugs and Devices and Cosmetics Act” and R.A. 7394 or the
“Consumer Act of the Philippines”, they still lack implementing guidelines
on the process of developing a probiotic product for use in aquaculture. Despite of this statutory deficit, probiotics are being used in a number of
aquaculture farms in the Philippines (Cruz et al., 2008; Moriarty, 1999). In
Negros Island, the use of probiotics is being combined with green water
technology in a tilapia-shrimp polyculture (Cruz et al., 2008). This system
Carlo C. Lazado et al. 104
is a current practice to reduce the expenses in using probiotics alone and
aeration which they stated as among the costliest inputs in shrimp culture in that region. The present authors tried to gather statistical information
from concerned agencies on the number of fish/shrimp farm in the
Philippines that are using probiotics as part of their production
management. Unfortunately, there are no existing reports that could be
provided at the moment. Even though probiotics are believed to be
“harmless”, having a specific set of guidelines on their application is a
must to avoid inappropriate use in the future and to ensure that they are
being applied as per their intent.
Figure 3. Modified diagram of the selection of probiotics for shrimp.
Mechanisms of probiotic actions in shrimp: Implications to tropical aquaculture 105
There are several readily available probiotics for aquaculture use in the
Philippine market. Biomin®, an international company locally operated by Ariela Marketing Co. Inc., has an array of probiotics and bioactive
compounds that are being marketed for the aquaculture industry
(www.biomin.net). For example, Aquastar® is a multispecies microorganism
cocktail with several published modes of action such as enhancement of
immune response, production of inhibitory substances and improvement of
water quality. BZT® Bio is a commercial cocktail of microencapsulated
bacteria and enzymes and is being marketed as having the capability of
reducing organic bottom solids and improving water quality
(www.atcvietnam.com.vn).
It is important to mention that there is no extensive probiotics research
in Philippine aquaculture. There is no identified research group that is
working on probiotics for aquaculture use unlike in other Asian countries such as Japan, Korea, Thailand and India. This observation is supported by a
limited number of published peer-reviewed papers on probiotics from
Philippine universities and research institutions.
The Scopus database (www.scopus.com) covers nearly 21,000 titles
from 5,000 publishers around the world. Searching the database using search
terms 1 (probiotics AND shrimp AND Philippines), search terms 2
(probiotics AND fish AND Philippines) and search terms 3 (probiotics AND
aquaculture AND Philippines) generated only 4, 3 and 1 document results
respectively (as of February 5, 2015). We acknowledge that there might be a
number of papers not covered in this quick literature survey or there are
some that are unpublished data. Despite the fact that probiotics are being used in Philippine aquaculture, this statistics reflects that its research
component is lagging behind hence should be incisively encouraged. A
concerted effort from the academe, the state and public sectors must be put
forward for the advancement and development of probiotics research in the
Philippines.
Nevertheless, it is also important to highlight some probiotic studies
conducted by Philippine-based researchers for they serve as foundation of
future studies. Torres et al. (1997) showed that commercial probiotics
demonstrated inhibitory activity against Philippine strains of pathogenic
Vibrio. In another study, the biocontrol property of commercial probiotics
against luminous Vibrio was demonstrated as well (Lio-Po et al., 2007). Trials
were also conducted in combining commercial probiotics and “green water” technology in shrimp and the results showed promise yet did not develop into
a full scale technology (Corre Jr et al., 2000). Janeo and Corre (2011)
demonstrated the effects of commercial probiotics in shrimp reared in a
bioaugmented system. Recently, a group from the University of the
Carlo C. Lazado et al. 106
Philippines Visayas demonstrated that applications of commercial probiotics
to saline tilapia could enhance growth, phytoplankton production and water quality (Mohamed et al., 2013). These data clearly show that Philippine-based
researchers are inclined to the use of commercial probiotics. To our
knowledge, there are no published reports characterizing and discussing the
probiotic potential of a microbial candidate isolated from the Philippines for
aquaculture. In the future, this has to be considered by Philippine-based
researchers with interest on probiotics for aquaculture because publications
from other countries have clearly demonstrated the potential of developing
probiotics from the indigenous microbiota of aquaculture species (Balcázar
et al., 2006;Lazado et al., 2015; Lazado & Caipang, 2014a; Martinez Cruz
et al., 2012; Merrifield et al., 2010; Mohapatra et al., 2013).
Summary. Shrimp aquaculture is an important industry thus
management should not be only profitably sound but it should be
ecologically sustainable as well. The industry is taking the right direction in
reducing the use of antibiotics and promoting the adoption of eco-friendly
alternatives like probiotics as disease control agents. Probiotics is a group of
microorganisms possessing a number of beneficial features in promoting
better health status of shrimp. These beneficial features are conferred
through different mechanisms. Probiotics promote better health status by
modifying the rearing environment and by influencing host’s physiological
responses leading to improved growth and disease resistance. Our
understanding of probiotics in shrimp as a prophylactic measure is limited
and mostly speculative. The published reports discussing the mechanisms of
probiotic actions in shrimp corroborate this conclusion. In particular, the
status of probiotics research and application in the Philippines partly
represents a microcosm of the global position of probiotics in shrimp culture.
There are numerous issues that need to be addressed particularly in research
approaches and legislations. Thus, several initiatives must be upheld in
response to the present knowledge gap. The promise of probiotics for an
effective and sustainable shrimp culture is enormous. Therefore, documented
mechanistic understanding on how these microorganisms deliver their
benefits is a must as this will be pivotal in their advancement as potential,
valuable and compelling alternative to antibiotics.
Acknowledgements
The Technical University of Denmark (Hirtshals, Denmark) is
acknowledged for the logistic assistance given to C.C. Lazado in writing this
paper.
Mechanisms of probiotic actions in shrimp: Implications to tropical aquaculture 107
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