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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


(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;


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


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).


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


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.


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


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).


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


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.


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


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


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


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.


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


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.


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