probiotics in shrimp aquaculture: avenues and challenges

Critical Reviews in Microbiology, 2009; 35(1): 43–66

R e v i e w A R t i c l e

Probiotics in shrimp aquaculture: Avenues and challenges

A.S. Ninawe1, and Joseph Selvin2

1Department of Biotechnology, Ministry of Science and Technology, New Delhi, India and 2Marine Bioprospecting Laboratory, Department of Microbiology, Bharathidasan University, Tiruchirappalli, India

Address for Correspondence: Joseph Selvin, Marine Bioprospecting Laboratory, Department of Microbiology, Bharathidasan University, Tiruchirappalli 620 024, India E-mail: [email protected]

(Received 18 June 2008; revised 18 Septmber 2008; accepted 5 December 2008)

Introduction

Viral and bacterial epizootics are considered as major limiting factor and constraints for the successful devel-opment and continuation of shrimp aquaculture produc-tion in terms of quality, quantity and regularity (Bachere et al. 1995; Mialhe et al. 1995; Selvin and Lipton 2003). Albeit disease management is an inherent component of any intensive animal production system, control-ling / preventing outbreaks in the aquatic environment is further complicated due to the inhabited interaction that exists between pathogens and their host particu-larly in the ‘in captivity’ production systems (Olafsen 2001). Broad spectrum antimicrobials have extensively been used as a means of reactive disease management strategy in aquaculture facilities. Considering the high promising results obtained in the in vitro screening of

commercial antibiotics, the post-infection therapy using antibiotics remain the method of choice for many farm-ers (Gram et al. 2001; Selvin and Lipton 2003a; 2004). However, excessive antibiotic use can lead to the emer-gence of bacterial resistance (Verschuere et al. 2000). Use of antibiotics and their consequences in shrimp farming has received attention from public health point of view due to the potential exposure of human consumers to antibiotic residues. The epidemiological evidences of food borne diseases suggests that fish/shrimp harvested from open seas are generally regarded as safe whereas those products from aquaculture were associated with food safety issues such as the risk of contamination with chemical and biological agents (Reilly et al. 1998). In addition, antibiotic application seems to be hazardous to human health, due to the development of clinically important resistant bacterial strains and possible failure

ISSN 1040-841X print/ISSN 1549-7828 online © 2009 Informa UK LtdDOI: 10.1080/10408410802667202

AbstractAs an alternative strategy to antibiotic use in aquatic disease management, probiotics have recently attracted extensive attention in aquaculture. However, the use of terrestrial bacterial species as probiotics for aquaculture has had limited success, as bacterial strain characteristics are dependent upon the environ-ment in which they thrive. Therefore, isolating potential probiotic bacteria from the marine environment in which they grow optimally is a better approach. Bacteria that have been used successfully as probiot-ics belong to the genus Vibrio and Bacillus, and the species Thalassobacter utilis. Most researchers have isolated these probiotic strains from shrimp culture water, or from the intestine of different penaeid spe-cies. The use of probiotic bacteria, based on the principle of competitive exclusion, and the use of immu-nostimulants are two of the most promising preventive methods developed in the fight against diseases during the last few years. It also noticed that probiotic bacteria could produce some digestive enzymes, which might improve the digestion of shrimp, thus enhancing the ability of stress resistance and health of the shrimp. However, the probiotics in aquatic environment remain to be a controversial concept, as there was no authentic evidence / real environment demonstrations on the successful use of probiotics and their mechanisms of action in vivo. The present review highlights the potential sources of probiotics, mechanism of action, diversity of probiotic microbes and challenges of probiotic usage in shrimp aquaculture.

Keywords: Shrimp-aquaculture; Probiotics; Antagonistic-microbes; Disease-management; Shrimp-disease; Antagonistic-bacteria

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44 A.S. Ninawe et al.

of antibiotic therapy (Shome and Shome 1999). As such, no approved antibiotics are available for shrimp farming, barring a few approved for controlling diseases in finfish farming. Therefore, novel antimicrobials with increased potency and least residual accumulation in shrimp tis-sue are required in lieu of conventional antibiotics for the management of bacterial epizootics.

Bacterial disease outbreaks particularly vibriosis and black shell disease impose a significant constraint on the production of fish and shellfish (Bachere et al. 1995; Verschuere et al. 2000; Selvin et al. 2005). The aquatic environment contains a plethora of opportunistic and secondary bacterial pathogens as well as beneficial and non-pathogenic bacterial strains. In hatchery and rear-ing facilities, the environmental conditions (availability of iron, osmotic strength, oxygen levels, pH, water qual-ity and temperature) and sometimes poor management practices (inadequate nutrition, overcrowding and overfeeding) can cause stress to the organisms being cultured and thus make them more susceptible to dis-ease outbreaks (Hansen and Olafsen 1999; Verschuere et al. 2000; Winton 2001). In addition, bacteria are easily introduced through natural or artificial food sources, inlet water and less frequently through vertical trans-mission from brood-stock (Alabi et al. 1997; Hansen and Olafsen 1999; Sandaa et al. 2003; Winton 2001). Although the traditional approach to rearing fish and shellfish is to create as clean and pathogen-free environment as pos-sible by treating water sources with filters, ozone and/or UV, total sterility of water cannot be achieved through pre-treatment (Alabi et al. 1997; Holstrom and Kjelleberg 1999). Furthermore, the loss of a stable microbial bal-ance through disinfection leaves an environment wide open for the proliferation of any opportunistic bacteria that make it through the system, hence limiting the natural biological control of opportunistic pathogens (Olafsen 2001).

Microorganisms have a critical role in aquaculture systems because water quality and disease control are directly related and closely affected by microbial activ-ity (Pillay 1992). Intensive cultivation systems obviously led to a change in the composition of environment and indigenous protective flora of the cultured organisms. This leads to an increase in the susceptibility of the host animal to opportunistic / secondary pathogens as well as reduced feed conversion ratio due to the imbalanced microbiota in the intestinal tract. However, it has been reported that both health and survival of organisms in intensive rearing systems could be improved substan-tially by manipulating the gut / environmental micro-biota with probiotic microorganisms and/or prebiotics, which can be added to the diet and/or to the environ-ment to promote the growth of beneficial bacteria in the gastrointestinal tract of the animal as well as in the detritivorous microbes in the pond bottom (Olafsen

2001; Lin 1995; Rengpipat et al. 1998a,b). The use of probiotics for disease prevention and improved nutri-tion in aquaculture is becoming increasingly popular due to an increasing demand for environment-friendly aquaculture. There have been many studies involving probiotics for use in aquaculture (e.g., Moriarty 1998; Gatesoupe 1994; Gram et al. 1999; Nikoskelainen et al. 2001; Panigrahi et al. 2004; Salinas et al. 2005), but the mode of action is incompletely understood. However, it is widely accepted that the mechanism of probiotics include inhibitory interaction (antagonism), production of inhibitory compounds, competition for chemicals and adhesion sites, improving the microbial balance, immune modulation and stimulation, and bioreme-diation of accumulated organic lead in the pond bottom (Lin 1995; Rengpipat et al. 1998; McCracken and Gaskins 1999; Verschuere et al. 2000).

The number of scientific publications on probiotics has doubled in the past three years and this recent inter-est (Kohn 2004) has been further stimulated by several factors including (1) exciting scientific and clinical find-ings using well documented probiotic organisms, (2) concerns over limitations and side effects of pharmaceu-tical agents, (3) consumer demand for natural products, in general, and (4) very promising aquatic antagonists for ecological aquaculture, in particular. All this has led to predictions of a tripling in sales by 2010 (European and US Probiotics Market research, 6 August 2003; www.frost.com). The key to the future of probiotics will be the establishment of a consensus on product regulation, including enforcement of guidelines and standards, appropriate efficacy studies that define strengths and limitations of products, and basic science studies that uncover the mechanisms of action of strains.

Survey of literature revealed that the status of probiot-ics in aquaculture has recently been reviewed on many angles particularly scope of applications in shrimp larviculture and mechanisms of action. The increasing number of reviews on probiotics in aquaculture ulti-mately enlightens the importance of probiotics on the development of aquaculture practices as environmental friendly and economical. In order to highlight the sig-nificance and need of present review on probiotics in shrimp aquaculture, brief contents of recent reviews on probiotics in aquaculture are introduced here to reveal the focus of available reviews. Tinh et al. (2008) reviewed functional role of probiotics in shrimp larviculture. This article reviews the current knowledge of in vivo mecha-nisms of action of probiotics in shrimp larviculture. The article highlighted the need of focused research on in vivo mechanisms of action including studies on gut microbiota composition, the use of gnotobiotic animals as test models, and the application of molecular tech-niques to study host-microbe and microbe-microbe interactions. The probiotics in marine larviculture has

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Probiotics in shrimp aquaculture: Avenues and challenges 45

also been reviewed by Vine et al. (2006) focusing on screening methods, probiont suitability and need of cost-benefit analysis. Decam et al. (2008) reviewed field data from Asia and Latin America on probiotic application in shrimp larviculture. This review exclusively focused on probiotic application of Bacillus strains in shrimp farming. The performance of a commercially available mixture of Bacillus strains (SANOLIFE® MIC) was evalu-ated using data from Asian and Latin-American hatch-eries, with P. monodon and L. vannamei. Watson et al. (2008) reviewed the status of probiotics in aquaculture focusing on mode of action and screening methods. However, scope of the review was mostly confined to mollusk culture. Wang et al. (2008) highlighted the need of effective preparation and safety evaluation of probi-otics. This review provided the perspective of digestive tract, thereby taking into account the experiences in introducing the preparation, use and safety evaluation of probiotics in aquaculture.

The available reviews are mostly confined to prospec-tive applications of probiotics in shrimp larviculture, particularly strains and / or mechanisms of action of probiotics. Literature is not available on the status of shrimp probiotics reviewed in multiple angles as to understand the prospects and challenges of probiotic development holistically. Therefore the present review is intended to bring out the avenue and challenges of probiotics in shrimp aquaculture particularly; inno-vative methods in probiotic development, regulatory aspects, potential strains, mechanisms of actions in vitro and in vivo, field realities and considerations for future research are reviewed and commented wherever neces-sary. This review would be the first report indicating the potential avenues of sponge associated marine bacteria as novel source for probiotic development. Albeit the present review aims to bring out the potentials and chal-lenges of probiotic development exclusively for shrimp aquaculture, the predictive/possible mode of action and potential strains developed in fish models are discussed wherever necessary to highlight the need of new lines of research in shrimp probiotics.

Probiotics: definitions

The term probiotic, meaning “for life,” is derived from the Greek language “pro” and “bios” (Gismondo et al. 1999). It was first used by Lilly and Stillwell (1965) in 1965 to describe “substances secreted by one microor-ganism which stimulates the growth of another”. The term ‘probiotic’ was originally coined as an antonym of antibiotics, since probiotics are beneficial microorgan-isms selectively proliferate to exclude competitively the harmful microbes. It may be because of this positive and general claim of definition that the term probiotic was subsequently applied to other subjects and gained a more

general meaning. In 1971, Sperti (1971) applied the term to tissue extracts that stimulate microbial growth. Parker (1974) was the first to use the term probiotic in the sense that it is used today. He defined probiotics as “organisms and substances which contribute to intestinal microbial balance.” Retaining the word substances in Parker’s defi-nition of probiotics resulted in a wide connotation that included antibiotics. In 1989, Fuller (1989) attempted to improve Parker’s definition of probiotic with the fol-lowing distinction: “A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance.” Fuller’s definition was a revision of the original probiotic concept which referred to protozoans producing substances that stimulated other protozoans (Lilly and Stillwell 1965). This revised definition emphasizes the requirement of viability for probiotics and introduces the aspect of a beneficial effect on the host, which was, according to his defini-tion, an animal. This definition is still widely referred to, despite continual contention with regard to the correct definition of the term. In 1992, Havenaar et al (1992) broadened the definition of probiotics with respect to host and habitat of the microbiota as follows: “A viable mono- or mixed culture of microorganisms which applied to animal or man, beneficially affects the host by improving the properties of the indigenous microbiota.” Salminen (1996) and Schaafsma (1996) broadened the definition of probiotics even further by no longer lim-iting the proposed health effects to influences on the indigenous microbiota. According to Salminen, a probi-otic is “a live microbial culture or cultured dairy product which beneficially influences the health and nutrition of the host.” According to Schaafsma, “Oral probiotics are living microorganisms which upon ingestion in certain numbers, exert health effects beyond inherent basic nutrition.” Current probiotic applications and scientific data on mechanisms of action indicate that non-viable microbial components act in a beneficial manner and this benefit is not limited just to the intestinal region (Salminen et al. 1999). The concept of probiotic activity has its origin in the knowledge that active modulation of the gastrointestinal tract (GIT) could confer antagonism against pathogens, help development of the immune system, provide nutritional benefits and assist the intestinal mucosal barrier (Vaughan et al. 2002). A joint action committee of Food and Agriculture Organization and World Health Organization defined probiotic as “live microorganisms, conferring a healthy benefit on the host when being consumed in adequate amounts” (FAO/WHO 2001).

The term “prebiotic” was introduced by Gibson and Roberfroid (1995) who exchanged “pro” for “pre,” which means “before” or “for.” They defined prebiotics as “a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or

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activity of one or a limited number of bacteria in the colon.” This definition more or less overlaps with the definition of dietary fiber, with the exception of its selec-tivity for certain species. The term “symbiotic” is used when a product contains both probiotics and prebiotics (Schrezenmeir and de Vrese 2001). Because the word alludes to synergism, this term should be reserved for products in which the prebiotic compound selectively favors the probiotic compound. In this strict sense, a product containing oligofructose and probiotic bifido-bacteria would fulfill the definition, whereas a product containing oligofructose and a probiotic Lactobacillus casei strain would not.

In aquaculture systems, the interaction between the microbiota and the host is not limited to the intestinal tract. Considering the complex ecological interactions in the aquatic environment, Moriarty (1999) suggested that the definition of a probiotic in aquaculture should include the addition of live autochthonous (naturally occurring) bacteria to tanks and ponds in which animals live. Therefore, Verschuere et al. (2000) have proposed a new definition, which allows a broader application of the term ‘probiotic’ in aquaculture. A probiotic is defined as “a live microbial adjunct that 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 toward disease, or by improving the qual-ity of its ambient environment.” Probiotics for aquatic organisms have been defined as “microbial cells that are administered in such a way as to enter the gastrointesti-nal tract and to be kept alive, with the aim of improving health” (Gatesoupe 1999).

Brief history of probiotics

There is a long history of health claims concerning liv-ing microorganisms in food, particularly lactic acid bacteria. Persian version of the Old Testament (Genesis 18:8) states that “Abraham owed his longevity to the consumption of sour milk.” In 76 B.C. the Roman his-torian Plinius recommended the administration of fermented milk products for treating gastroenteritis (Bottazzi 1983). Since the advent of the microbiology era, some investigators including Carre (1887), Tissier (1984), and Metchnikoff (1907) attributed such health effects to shifts of the intestinal microbial balance. Metchnikoff (1907) claimed that the intake of yogurt containing Lactobacilli results in a reduction of toxin-producing bacteria in the gut and that this increases the longevity of the host. Tissier (1984) recommended the administration of Bifidobacteria to infants suffering from diarrhea, claiming that Bifidobacteria supersede the putrefactive bacteria that cause the disease (1984). He showed that Bifidobacteria were predominant in

the gut microbiota of breast-fed infants. Administration of probiotics to animals started in the 1920’s and the name was introduced in the 1970s to describe microbial feed supplements given to both humans and animals (Berg 1998). The lactic acid bacteria have been widely used and researched for human and terrestrial animal purposes, and lactic acid bacteria are also known to be present in the intestine of healthy fish (Ringø and Gatesoupe 1998; Hagi et al. 2004). Although the use of probiotics in human and animals has been established and improved as technology for commercial and clinical practice, the use of probiotics in aquaculture has short history. In 1980, Yasuda and Taga (1980) predicted that bacteria would be found to be useful both as food and as biological control agents of fish disease and activators of the rate of nutrient regeneration in aquaculture. The pioneer studies on the screening of probiotic bacteria from the aquaculture environments were reported in the late 1980s (Dopazo et al. 1988; Kamei et al. 1988). Vibrio alginolyticus has been employed as a probiotic in many Ecuadoran shrimp hatcheries since late 1992 (Garriques and Arevalo 1995). As a result, hatchery down time was reduced from approximately 7 days per month to less than 21 days annually, while production volumes increased by 35%. The overall antibiotic use was decreased by 94% between 1991 and 1994. The addi-tion of probiotics is a common practice in commercial shrimp hatcheries in Mexico (Rico-Mora et al. 1998). Competitive exclusion of potential pathogenic bacteria effectively reduces or eliminates the need for antibiotic prophylaxis in intensive larviculture systems (Garriques and Arevalo 1995).

Proactive disease management

The World Health Organization (WHO) recommended the preventative (prophylactic) approaches to disease management rather than costly post-effect treatments (WHO 2002). The increasing political and environmen-tal pressure to decrease the use of antibiotics and other therapeutic chemicals in agriculture and aquaculture has stimulated research into more environmentally friendly approaches for disease control (Hansen and Olafsen 1999; Verschuere et al. 2000). The common tool for proactive (disease prevention) is by incorporating compounds that stimulates the host’s immune system. The prophylactics are grouped into three categories, viz., nutrients which act indirectly on cell physiology, those that work in a specific manner such as vaccines, and those that are non-specific in nature such as the glucans, alginates and lipopolysaccharide (LPS)-based materials (Itami et al. 1989 1992 1998; Latchford el al. 1996; Karunasagar et al. 1996; Alabi et al. 1999; Chang et al. 2000; Neuman 2001;NG_BIB_000_053 Bachere 2003). The immune systems of shrimp are not developed as that

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of the vertebrates and no specific antibodies or proteins are produced in response to the structural components of a particular pathogen. Though mono and multivalent vaccines have been developed against several bacterial diseases in fish, such vaccine preparations are not suc-cessful at times in the case of shrimps. In addition, as new diseases and pathogens may emerge from time to time, practically it is impossible to develop proactive strategies using vaccines. But the shrimp immune system could be stimulated in a ‘non-specific’ manner using a variety of cell wall fragments as well as components from various microorganisms (Devaraja et al. 1998; Itami et al. 1998; Chang et al. 2000). These formulations are highly dose dependent (Raa et al. 1992) and therefore at times they are less successful. These lacunae, prompted to develop and test novel prophylactic antimicrobials from marine organisms so as to prevent the onset and spread of dis-ease throughout the culture period (Selvin and Lipton 2004; Selvin et al. 2004). Retrospective of work revealed that seaweeds (such as Ulva sp.) apart from forming food for herbivorous fish (Shipgel et al. 1998) also stimulate the defense system of fish by their alginates and polysac-charides (Nordmo et al. 1995). The marine based natural products like spray-dried Tetraselmis suecica and extract of tunicate Ecteinescidia turbinata were experimentally proved of their respective role in managing fish dis-eases (Austin and Day 1990; Davis and Hayasaka 1984). Recently, the potential application of marine bioactive secondary metabolites in shrimp disease management has been realized. Shrimp disease management using bioactive marine secondary metabolites was emerged as an eco-friendly approach (Selvin and Lipton 2003,

2004a). The secondary metabolites isolated from green alga Ulva fasciata and a marine sponge Dendrilla nigra were developed as antimicrobial agents for the control of bacterial diseases (Selvin and Lipton 2004; Selvin et al. 2004). Seaweeds based products are being evaluated for the development of proactive management tools (Selvin et al. 2008, unpublished data; Huang et al. 2006). The sustainability of production is dependent on the equilibrium between the environmental qualities, the disease prevention by prophylactics, epidemiological surveys of the pathogens, and the health status of the shrimp. Therefore, the prevention and the control of shrimp diseases (management) are emerging as an integrated approach. Considering the potential applica-tion of probiotics in animal husbandry, the antagonistic probiotics are being developed as a package of practice for the proactive management of mid-culture outbreaks, shrimp health and growth rate and maintenance of water and soil quality holistically. Albeit the potential of probiotics has been well established, the constraints in probiotic development need to be considered in the evaluation of novel as well as commercially available probiotics (Figure 1).

Evaluation of probiotics

The use of probiotic bacteria, based on the principle of competitive exclusion, and the use of immunos-timulants are two of the most promising preventive methods developed in the fight against diseases during the last few years (Verschuere 2000). One of the main challenges in developing probiotic bacteria is using

Shrimp

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Constraints of probiotics Potentials of probiotics

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Figure 1. Potentials and constraints of probiotics in shrimp aquaculture.

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appropriate selection and colonization methods. The selection criteria for probiotic bacteria should evaluate the colonization methods, competition ability against pathogens and the immunostimulatory growth effect on shrimp (Gatesoupe 1999; Gomez-Gil et al. 2000). By applying these bacteria in shrimp farms, a biological equilibrium between competing beneficial and del-eterious microorganisms could be produced. In fishes, it has been established that probiotic bacteria could produce some digestive enzymes, which might improve the digestion of fish, thus enhancing the ability of stress resistance and health of rainbow trout, Oncorhynchus mykiss (Irianto and Austin 2003). Further, probiotic bac-teria directly uptake or decompose the organic matter or toxic material in the water and sediment thereby improv-ing the quality of water (Wang et al. 2005). According to the findings of Chinese researchers, when these bacteria were added into the water, they could decompose the excreta of shrimps, remaining food materials, remains of the plankton and other organic materials to CO

2, nitrate,

and phosphate. In recent years, culture-independent molecular methods have been developed to evaluate the potential probiotics (Vergin et al. 2001). These include pulse field gel electrophoresis (PFGE), RFLP of 16S rRNA gene, multiplex PCR, Arbitrary Primed (AP) PCR, random amplified polymorphic DNA (RAPD), denatur-ing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), and/or terminal restriction fragment length polymorphism (TRFLP) (Pond et al. 2006).

The selection and application technologies of probi-otics must be based on thorough understanding of the mechanisms involved and the putative consequences. An essential part of that understanding can be provided by studies looking in detail at host-microbial interac-tions. A key experimental strategy to study these interac-tions is to first define the functioning of the host in the absence of bacteria and then to evaluate the effects of adding a single or defined population of microbes, or certain compounds (i.e., under axenic or gnotobiotic conditions) (Gordon and Pesti 1971). Marques et al. (2004a,b; 2005) have previously shown that the brine shrimp Artemia is instrumental in the development of a gnotobiotic test system for studying the effect of food composition on survival and growth in the presence or absence of a challenge test. The test system uses a gnoto-biotic Artemia culture in which the microbial community is totally controlled (Marques et al. 2006). The Artemia is essential part of the live food chain for the culture of fish and shell fish larvae (Sorgeloos et al. 1986). Artemia is a continuous and non-selective particle filter feeder able to consume a maximum particle size of 25–30 µm for nau-plii and 50 µm for adults. Considering the wide variety of non-selective feeding habit of Artemia, it can be used to encapsulate (bioencapsulation) potential probiotics

such as baker’s yeast and microalgae (Dobbeleir et al. 1980; Sorgeloos et al. 1986). According to Marques et al. (2005), the performance of Artemia fed with poor-qual-ity feeds can be easily improved by the addition of non-pathogenic bacteria, partly because they are used as a source of nutrients, while the effects of such bacteria are less pronounced in animals fed with better quality feeds. The use of dead bacteria (especially when added once) was not as efficient as the feeding condition to control diseases in gnotobiotic Artemia (Marques et al. 2006). The method used to obtain dead bacteria (autoclaving) could have contributed to this result, as this method has a strong potential to destroy several compounds, like vita-mins, proteins, and fatty acids, and to induce damages to cell membrane lipids (Marques et al. 2004a). Therefore, future studies should include less potentially destructive methods such as formalin inactivation and UV exposure to inactivate bacteria. Fermented dairy products are generally considered to be one of the most suitable vehi-cles for the administration of an adequate number of probiotic bacteria to the consumer (Casteele et al. 2006). The development of suitable vehicles / delivery mecha-nism for the effective introduction of probiotics in the shrimp gut is inevitable for the attainment of successful probiotics in the “in captivity” environments. Although still a matter of debate, several authors have indicated that a minimal concentration of 1 × 106 (colony forming units) cfu g–1 of a product is required to exert a probiotic effect (Ravula and Shah 1998; Shah 2000; Vinderola and Reinheimer 2000; Roy 2001; Talwalker and Kailasapathy 2004) in higher animals.

Problems in probiotics development

As probiotics are highly sensitive to many environmen-tal factors, development of fermentation technologies requires new approaches. To date, probiotic produc-tion has almost exclusively been carried out using conventional batch fermentation and suspended cul-tures (Lacroix and Yildirim 2007). However, the strains developed through novel cultivation approaches require strategic process optimization. At the species level, misi-dentification was mostly situated in the taxonomically complex genera Lactobacillus and Bifidobacterium (Huys et al. 2006). Based on the finding that more than 28% of commercial cultures intended for probiotic use were misidentified at the genus or species level, it is reasonable to assume that deficiencies in the microbiological qual-ity and label correctness of probiotic products reported by several authors (Yeung et al. 2002; Fasoli et al. 2003; Temmerman et al. 2003; Drisko et al. 2005; Masco et al. 2005) may be largely due to the incorporation of incor-rectly identified bacterial cultures. It has been widely accepted that 16S rDNA sequencing analysis may be universally regarded as the best tool for the taxonomic

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positioning of probiotic cultures. However, it should be kept in mind that 16S rDNA sequences have a limited resolution for the discrimination of several closely related lactic acid bacterial species used in probiotic production (Hong et al. 2005). In most cases, the terrestrial derived probiotic strains are seldom successful in shrimp aqua-culture, due to their lack of competency in aquatic and/or marine environment. Further, most of the available reports focused on the potential probiotic strains which are readily cultivable with available media. But it has been reported that the marine bacterial endosymbionts are uncultivable with available media (Selvin et al. 2004). In principle, the endosymbiotic antagonistic microbes might have the potential probiotic efficiency, since they are established antagonistic symbionts of marine inver-tebrates. Therefore, exploring the marine bacterial endo-symbionts would be a prospective approach for develop-ing novel shrimp probiotics.

Guidelines and Regulations

A number of definitions for the term ‘probiotic’ have been used over the years but the one derived by the Food and Agriculture Organization of the United Nations-World Health Organization (FAO-WHO) (FAO/WHO 2001) are endorsed by the International Scientific Association for Probiotics and Prebiotics (Reid et al. 2003). As per the FAO-WHO guidelines, the scope of probiotics could be defined as “live microorganisms, which when adminis-tered in adequate amounts, confer a health benefit on the host”. This definition retains the historical elements for the use of living organisms for health purposes but does not restrict the application of the term only to oral probiotics with intestinal outcomes (Reid and Bruce 2006). The guidelines that stipulate what is required for a product to be called a probiotic were published by FAO-WHO in 2002 (FAO/WHO Guidelines 2002). The requirements include, the strains must be designated individually, speciated appropriately and should retain a viable count at the end of their shelf life in the desig-nated product formulation that confers a proven clinical end-point. Although member nations were encouraged to use these guidelines, the fact that some products con-tinue to be of dubious quality and claim health benefits that are not supported by appropriate, peer-reviewed human studies suggests that many regulatory authori-ties are not yet aligned (Canganella 1997; Temmerman et al. 2003; Huff 2004). The selection and evaluation of potential probiotic candidates is a multistep proc-ess focusing on functional, safety, and technological aspects (Sanders and Huis in’t Veld 1999; Saarela et al. 2000; Reid 2006). There is a growing awareness that the correct identification of a probiotic strain is one of the first prerequisites documenting its microbiological safety (Huys et al. 2006). Documentations of proven

clinical efficacy and known mechanisms of action in addition to clearly outlined dosage, duration of use, and safety parameters will enable caregivers to recommend products and enable consumers to purchase probiotic foods and over-the-counter products with a high level of confidence. However, the systemic studies on the efficacy, existence and transmission risks of probiot-ics in aquaculture were not reported. As new probiot-ics emerge alongside genetically modified organisms (GMOs) that are designed specifically to treat disease (Steidler et al. 2003), long-term monitoring will be important to ensure that safety issues and (in the case of GMOs) proper environmental containment issues are addressed. According to Henriksson et al. (2005), more clinical studies need to be performed, preferably com-paring one probiotic product against another or against standard medical practice. In this way, the strengths and limitations of probiotics can be determined. Therefore, holistic evaluations of newly developed probiotics are to be conducted in well-designed field trails to validate / commercialize probiotic products in aquaculture. But there is no regulatory guidelines set for the usage of pro-biotics in aquaculture. Considering the emerging issues on the transfer of zoonoses from aquaculture systems and possible risk of transferring the potential probiotic strains to pathogens, the usage of terrestrial and dairy strains aquaculture need to be monitored and regulated with appropriate guidelines.

Potential sources of antagonistic probiotics

In agriculture, the use of antagonistic bacterial strains that control the concentrations of potential pathogens by competitive exclusion has been successful in pre-venting disease outbreaks in many species (Vanbelle et al. 1990; Nisbet et al. 1994; Corrier et al. 1995a, b; Hansen and Olafsen 1999). However, the use of terrestrial strains of bacteria as probiotics for aquaculture has had lim-ited success, as strain characteristics of bacteria are dependent upon the environment in which they thrive. Therefore, isolating potential probiotic bacteria from the marine environment in which they grow optimally is a better approach.

Gram-negative bacteria are considered to cause the majority of bacterial problems associated with shrimp diseases (Bachere et al. 1995; Verschuere et al. 2000; Selvin et al. 2005). With the exception of terrestrially derived Gram positive bacteria particularly lactic acid bacteria, the selection of probiotics in marine aquacul-ture is dominated by Gram-negative species (Austin et al. 1995; Gibson et al. 1998; Gram et al. 1999; Spanggaard et al. 2001). The selection of Gram-negatives is probably due to the suitability of their r-selected strategy, which provides initial, rapid, direct competition, i.e., for nutri-ents, attachment, etc., with the potentially harmful,

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opportunistic or pathogenic bacteria which are also fast-growing (Vine et al. 2006). As the aquatic culture environment stabilizes and matures, the microbiota may become a community dominated by K-selected species (Salvesen et al. 1999). Although r-selected pro-bionts may appear suitable during the early stages of larval rearing, the potential benefits of K-selected spe-cies should be considered if a long-term probiotic treat-ment is required. This problem of strain degeneration is important when considering Gram-negative candidate probionts as unlike some Gram-positive bacteria, they are not endospore-forming. Therefore to maintain viable cultures, Gram-negative candidate probionts require more frequent sub-culturing on solid or liquid media. Lyophilization, immobilization techniques, and biomass production under fermentation conditions are considered to be the reliable and economical methods of Gram negative bacterial production/application in shrimp aquaculture.

In a review of the bacterial flora associated with cold water marine fish eggs and larvae, Hansen and Olafsen (1999) showed that the highly diverse bacterial flora on fish eggs functions as a protective barrier against poten-tial pathogens, and that these bacteria is established in the intestinal flora in juvenile fish through the uptake of water for osmoregulation. Similarly, they suggested that the gut microbiota of adult fish would be greatly influ-enced by these pioneer bacteria found in the diet and ambient water of the developing larvae thus, stimulating the search for bacteria that would inhibit the coloniza-tion of pathogenic bacteria in marine fish eggs.

In general, the bacterial strains which are non-hemo-lytic, normal microbiota of the habitat, forms swarming with other non-pathogenic strains, and produce inhibi-tory substances would be the most preferred candidates for probiotics (Farzanfar 2006). As in nature, a health balance of microorganisms in the ponds should include bacteria that thrive in very specific environmental niches as well as those that thrive in more general niches (Jöborn et al. 1997). As beneficial bacteria are present in the farm environment and may be concentrated from the feed and surrounding water into the intestine of healthy animals, it is reasonable that the isolation and development of probiotics could start with an initial survey of the bacterial community in rearing water when disease is absent and include species found in the intestinal tract of healthy individual and the seawater in which the cultured animals grow (Vine et al. 2006). Using this information and further experiments on the probiotic effects of specific bacterial strains, it should be possible to establish a healthy balance of bacterial spe-cies by preemptively colonizing the treated rearing water with known probiotic organisms rather than allowing the accidental colonization by unknown and poten-tially opportunistic bacterial pathogens (Verschuere et

al. 2000). Several studies have reported that the rate of isolation of useful probiotic ranges from 1% to 4% of the total number of screened bacteria (Pybus et at. 1994; Spanggaard et al. 2001). With such low recovery num-bers, there is a need to screen several hundred bacterial strains in order to obtain sufficient numbers of potential probiotics for further short listing.

The marine environment has been mined for novel microorganisms used in drug development (Selvin et al. 2004) and is likely the largest contributor of bacteria to the aquaculture. The research is being under progress to establish sponge associated marine bacteria as poten-tial source of novel shrimp probiotics (Selvin et al. 2008, unpublished data). It was found that the endosymbiotic marine actinobacterium Nocardiopsis alba MSA10 iso-lated from the marine sponge Fasciospongia cavernosa showed potential antagonistic activity against promi-nent Vibrio pathogens of Penaeus monodon in vitro and in vivo. Sfanos et al. (2005) made a survey of bacterial samples isolated from wild marine sources includ-ing macroalgae, seawater, and sea sediment to screen potential probionts. Numerous bacterial community have been explored from unique marine environments, such as hydrothermal vents (Jeanthon 2000), marine sea sediments (Cifuentes et al. 2000; Llobet-Brossa et al. 1998), marine biofilms, microalgae blooms (Seibold et al. 2001), and marine sponges (Lafi et al. 2005). The rep-resentation of bacterial groups uncovered were mostly gamma proteobacteria tending to dominate in most communities (Eilers et al. 2000). Recently marine bacte-rial endosymbionts are emerging as potential source for the development of novel probiotics (Selvin et al. 2004: Selvin et al. 2008, unpublished data).

It has been considered that the digestive tract of healthy individuals is a primary source for potential probionts as these bacteria would be pre-conditioned to thrive in high acid environments and helps to exclude pathogens from adhesion sites in the intestinal wall (Verschuere et al. 2000). Beneficial bacteria isolated from the abalone digestive tract enhanced the growth (8–33%) and survival rate (2.5 times) of abalone follow-ing infection with V. anguillarum (Macey and Coyne 2005). Three nonhemolytic species, Bacillus sp. (most similar to Bacillus psychrodurans), P. carotinifaciens (an astaxamthin-producing bacteria), and S. mamurdoensis (a new species originally isolated from a cyanobacterial mat in an Antartic pond) specific to the intestinal tract of healthy adult sablefish carry potential probiotic charac-ters (Reddy et al. 2003).

Bacteria in the aquatic environment and certainly those in the diet influence the composition of the fish intestinal tract where they can positively affect the health of the organism (Verschuere et al. 2000). In addi-tion to gut microbiota which can exclude the adhesion of other species to the intestinal wall, bacteria that can

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out-compete pathogens for carbon and energy sources in the aquatic environment may also be good candi-dates for probiotic mixtures (Verschuere et al. 2000). Furthermore, microorganisms displaying antibacterial properties have often been discovered associated with macroalgae and other sources in the marine environ-ment (Sfanos et al. 2005), and these offer a third source of potential probionts. Literature evidenced that a wide variety of probiotic strains could be developed from dif-ferent sources.

Successful and commercial probiotics in aquaculture

In parallel to the growth of probiotic application ranging from food supplements to biotherapeutics, the biodiver-sity of strains exhibiting potentially probiotic functional-ities has increased remarkably in recent years. The large majority of commercial probiotic products contain one or multiple strains of lactic acid bacteria primarily belong-ing to the genera Lactobacillus (Donkor et al. 2007; Geier et al. 2007), Bifidobacterium, Lactococcus, Pediococcus, Enterococcus, and Streptococcus. In addition, other bac-terial taxa such as Propionibacterium spp., Bacillus spp. and Escherichia coli and the yeast Saccharomyces boular-dii have also been used in probiotic products (Holzapfel et al. 1998; Klein et al. 1998; Mercenier et al. 2003). Some Bacillus sp. (B. megaterium, B. Polymyxa, B. subtilis, B. licheniformis), lactic acid bacteria (Lactobacillus sp., Carnobacterium sp., Streptococcus sp.), Pseudomonas sp. (P. fluorescens) and Vibrio sp. (V. alginolyticus, V. salmo-nicida-like) have been proposed and tested as probiotics in aquaculture (Gatesoupe 1991; Verschuere et al. 2000). Although studies have shown that lactic acid bacteria is effective in inhibiting the growth of various Vibrio spe-cies in Atlantic cod fry Gadus morhua (Gildberg et al. 1997) and turbot larvae (Gatesoupe 1994), the probiotic effects lasted only for a brief time after feeding was dis-continued. Lactic acid bacteria are known to produce growth inhibiting factors such as bacteriocins that are particularly useful against other Gram positive bacteria (Stoffels et al. 1992), however, since most of the known pathogens in aquaculture are Gram negative, and lactic acid bacteria account for only a small part of the gut microbiota of fish, their usefulness in aquaculture is debatable (Verschuere et al. 2000). Pseudoalteromonas have been found to synthesize biologically active com-pounds with antibacterial, algicidal, anti-algal, and bacteriolytic properties (Holstrom and Kjelleberg 1999). This relatively new genus has been exclusively isolated from marine environments throughout the world (Enger et al. 1987), and species within this genus are often found associated with eukaryotes (Holstrom and Kjelleberg 1999). One isolate has even produced a bactericidal antibiotic against methicillin-resistant Staphylococcus aureus (Isnansetyo and Kamei 2003). Maeda et al. (1997)

showed that the addition of an antimicrobial strain of Pseudoalteromonas undina repressed the growth of pathogenic bacteria and viruses in fish and crustacean farming.

Four species including, B. pumilus, Micrococcus luteus, P. fluorescens and P. putida are currently included in bacterial mixtures that are marketed as probiotics for aquaculture (Prowins Biotech Private Ltd., India). Additionally, Bacillus sp. have been successfully used as probiotics in the aquaculture of black tiger shrimp (Penaeus monodon) in Thailand, where there was an improvement in the growth rate (47%) and survival rate when challenged with Vibrio harveyi (Rengpipat et al. 1998). Aeromonas media UTS strain A199 has been shown to be a potential probiotic for the management of bacterial (Gibson et al. 1998; Tan et al. 2003) and fungal pathogens (Lategan and Gibson 2003; Lategan et al. 2004a,b) in the aquaculture industry (Lategan et al. 2006). Pseudomonas fluorescens (AH2) was shown to be strongly inhibitory against Vibrio anguillarum in model systems and it was found that this effect could be trans-ferred to an in vivo situation where the mortality rate in rainbow trout infected with V. anguillarum was sig-nificantly reduced by the addition of the probiotic bac-terium to the tank water (Gram et al. 1999). Rengpipat et al. (2000) showed that the survival and growth of the black tiger shrimp (Penaeus monodon), fed with probi-ont Bacillus S11, was increased when compared with non-treated shrimp. The addition of bacterium CA2 as a food supplement to auxenic cultures of Crassostrea gigas larvae was found to consistently enhance the growth of the oyster larvae regardless of the season of the year (Douillet and Langdon 1994). Thus, probiotics have been shown to be effective in a wide range of species for the promotion of growth, enhanced nutrition, immunity and survival. A partial list of prospective probiotics in aquaculture is presented in Table 1 Some of the selective strains widely used / evaluated for probiotic application in aquaculture are given below.

Bacillus

With the expansion of the commercial farming of Penaeid shrimp and the increased intensication of culture systems, there is a growing demand for well-balanced, nutritionally complete and cost-effective for-mulated feeds (Linan-Cabello et al. 2002). For several years, there has been continuing interest in identifying alternatives to shmeal for use within aqua feeds (Tacon et al. 1998; FAO 2002; New and Wijkstrom 2002; Naylor et al. 2000). Among the ingredients being investigated as alternatives to shrimp meal and soybean products (Glycine max) have been some of the most promising (Storebakken et al. 2000; Swick 2002), because of the security of supply, price and composition (Cain and

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Garling 1995). The probiotic Bacillus sp. increased the digestibility of the vegetable ingredients used as alternatives to shmeal in shrimp diets (Solano and Soto 2006). The genus Bacillus constitutes a diverse group of rod-shaped, Gram-positive bacteria, char-acterized by their ability to produce a robust spore. Most Bacillus species are not harmful to mammals, including humans and are commercially important as producers of a high and diverse amount of secondary

metabolites including antibiotics, bio-insecticides, biosurfactants and enzymes (Ferrari et al. 1993; Godfrey and West 1996; Olmos et al. 1996 1997 1998; Olmos 2003). Because Bacillus bacteria secrete many exoenzymes (Moriarty 1996 1998), these bacteria have been used widely as putative probiotics (Ochoa-Solano and Olmos-Soto 2006). Studies have shown that when these bacteria were administered as probiotics in the shrimp Penaeus monodon, growth and survival were

Table 1. Prospective probiotics evaluated for shrimp aquaculture applications.

Strain

Source

Evaluated for

Effective dose / mode of application

Reference

Bacillus S11 Black tiger shrimp Growth and survival of black tiger shrimp Penaeus monodon

1 kg wet wt (∼100 g dry wt) of BS11 (∼1010 CFU g–1) in 3 kg of feed (2.5% BS11/3 kg)

Rengpipat et al. 2003

Bacillus subtilis BT23 Shrimp culture ponds Against the growth of Vibrio harveyi isolated by agar antagonism assay from Penaeus monodon

106–108 CFU ml–1 for 6 d Vaseeharan and Ramasamy 2003

Pseudomonas sp. PM11Vibrio fluvialis PM17

Gut of farm reared sub-adult shrimp

Immunity indicators of Penaeus monodon

Pseudomonas sp. PM 11 @ 103 bacterial cells ml−1 for 3 days and V. fluvialis PM 17 @ 103 bacterial cells ml−1 for seven days

Alvandi et al. 2004

Arthrobacter XE-7 Isolated from Penaeus chinensis

Protection of Penaeus chinensis post-larvae from pathogenic vibrios such as Vibrio para-haemolyticus, Vibrio anguil-larum and Vibrio nereis

106 CFU/ml Li et al. 2006

Bacillus subtilis and B. megaterium

Marine environment Production of digestive enzymes proteases, carbohy-drolases and lipases

Potential application in shrimp feeds

Solano and Soto 2006

Paenibacillus spp., B. cereus and Pa. polymyxa

Seawater, sediment and marine fish-gut samples

Against pathogenic Vibrios 104 and 105 CFU ml–1 Ravi et al. 2007

Synechocystis MCCB 114 and 115

Seawater Antagonism against V. harveyi Post–larvae fed on the cyanobacterial cultures

Preetha et al. 2007

Bacillus licheniformis Shrimp pond Intestinal microbiota and immunity of the white shrimp Litopenaeus vannamei

B. licheniformis suspension of 105 CFU ml−1 for 40 days

Li et al. 2007

Lactic-acid bacteria Shrimp gut Survival of marine shrimp, Litopenaeus vannamei chal-lenged with V. harveyi

Liquid diet supplemented with B6 strain at 10(8) CFU/mL

Vieira et al.2007

Lactobacillus plantarum Shrimp isolate Immune response and micro-biota of shrimp digestive tract of Litopenaeus vannamei chal-lenged with V. alginolyticus and V. harveyi

1010 CFU /kg diet /108 CFU /kg feed

Chiu et al. 2007

Vibrio alginolyticus UTM 102, Bacillus subtilis UTM 126, Roseobacter gallaecien-sis SLV03, and Pseudomonas aestumarina SLV22

Gastrointestinal tract of adult shrimp Litopenaeus vannamei

Antagonism against the shrimp-pathogenic bacterium, Vibrio parahaemolyticus PS-017

Feed supplement Balcázar et al. 2007

Bacillus subtilis UTM 126 Shrimp culture pond Protection against vibriosis in juvenile Litopenaeus vannamei

105 CFU/g Balcázar and Rojas-Luna 2007

Pediococcus acidilactici Strain MA 18/5M, CNCM Survival of Litopenaeus styliros-tris against Vibriosis caused by Vibrio nigripulchritudo

Probiotic-coated pellet feed Mathieu et al. 2008

B. subtilis, B. natto, and B. licheniformis

Not available Growth and digestive enzyme activity of Litopenaeus vannamei

1.5 to 7.5% supplemented to the feed

Gómez and Shen 2008

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improved and immunity was enhanced (Rengpipat et al. 1998a,b 2000). However, the nutritional effects of probiont bacteria, especially the effects of the bacteria on digestive enzyme activity, have not been evalu-ated in aquaculture. Recently, Ziaei-Nejal et al. (2006) examined the effect of Bacillus probionts on digestive enzyme activity, survival and growth in the shrimp Fenneropenaeus indicus. Bacillus enzymes are very efficient in breaking down a large variety of carbohy-drates, lipids and proteins into smaller units. Bacillus species grow efficiently with very low-cost carbon and nitrogen sources (Sonnenschein et al. 1993). Bacillus species also degrade organic accumulation in ponds of shrimp cultures (Lin 1995; Rengpipat et al. 1998a,b; Verschuere et al. 2000). Probiotic Bacillus S11 in feed form has been proved to be beneficial to growth and survival of shrimp (Rengpipat et al. 1998a,b). An experiment on combined effect of ozone and probiotic diets showed that, it was possible to use ozone just for disinfection during the Vibrio challenge test without harming shrimp and probiotic bacteria in internal sys-tem of shrimp (Meunpol et al. 2000). Vasseharan and Ramasamy (2003) reported that pathogenic vibrios are controlled by a probiotic strain Bacillus subtilis BT23, under laboratory conditions. Kennedy et al. (1998) inoculated a strain of B. subtilis isolated from the com-mon snook Centropomus undecimalis into rearing water, which resulted in the apparent elimination of Vibrio spp. from the snook larvae. Taking into account the advantageous characteristics of Bacillus, these bac-teria are good candidates for consideration as probiot-ics in shrimp diets (Olmos-Soto et al. 2003).

Probiotic Bacillus offered an alternative to antibiotic therapy for sustainable aquaculture. There are severe problems with the use of antibiotics in aquaculture (Baticados et al. 1990). If antibiotics are used to kill bacteria, either strains of the pathogen or different bac-teria survive because they carry genes for resistance. These will then grow rapidly because their competitors are removed. Virulent pathogens that then re-enter the hatchery tank or aquarium, perhaps from within bio-films on water pipes or air lines or in the guts of the ani-mals where they were protected from the antibiotic, can then exchange genetic information with the resistant bacteria and survive further doses of antibiotic. Thus, antibiotic-resistant strains of the pathogen evolve very quickly. There are several reasons why it is better to add Bacillus rather than antibiotics to control Vibrio spe-cies. Many different antibiotic compounds are naturally produced by a range of Bacillus species (Moriarty 1998). Bacillus secretes many enzymes that degrade slime and biofilms and allow Bacillus and their antibiotics to penetrate slime layers around Gram negative bacteria. Furthermore, Bacillus competes for nutrients and thus inhibits other bacteria from growing rapidly. Thus any

resistant bacteria cannot multiply readily and transfer resistance genes (Hong et al. 2005). As there are many different mechanisms involved in the probiotic proc-ess of competition and exclusion, it is difficult for the pathogens to evolve all the necessary resistance genes together. Therefore, the presence of the Bacillus pro-biotic significantly improved shrimp survival in most treatments. Because, administration of the probiotic significantly changed the proportion of Bacillus bacteria in the gut flora, the increased survival by shrimp may be due to the exclusion of other bacteria (especially harm-ful bacteria) by the probiont, particularly in the larval and early postlarval stages where the Bacillus bacteria were dominant (Verschuere et al. 2000). In P. monodon, Bacillus used as a probiotic, was able to colonize both the culture water and the shrimp digestive tract; the Bacillus was able to replace Vibrio spp. in the gut of the shrimp, thereby increasing shrimp survival (Rengpipat et al. 1998a). Bacillus bacteria are able to out-compete other bacteria through the production of antibiotics (Moriarty 1998). Many different antibiotic compounds are produced naturally by a range of Bacillus species, and it appears that other bacteria would be unlikely to have resistance genes to all of the antibiotics produced by the Bacillus probionts, especially if they had not been exposed to the Bacillus previously (Moriarty 1998). Bacillus administration has also been shown to increase shrimp survival by enhancing resistance to pathogens by activating both cellular and humoral immune defenses in shrimp (Rengpipat et al. 2000). Bacillus surface anti-gens or their metabolites act as immunogens for shrimp by stimulating phagocytic activity of granulocytes (Itami et al. 1998). Administration of the Bacillus bacteria to shrimp resulted in an increase in the specific activity of lipase, protease and amylase in the shrimp’s diges-tive tract. Because gram-positive bacteria, particularly members of the genus Bacillus, do secrete a wide range of exoenzymes (Moriarty 1996 1998), sometimes it may not be possible to distinguish between activity due to enzyme synthesized by the shrimp and activity due to enzyme synthesized by bacteria.

Vibrio

Numerous Vibrio species have been reported as pathogenic bacteria to various penaeid shrimps (Chanratchakool et al. 1995; Alapide-Tendencia and Dureza 1997; Riquelme et al. 1997; Ringo and Vadstein 1998). Non-pathogenic strains of V. alginolyticus have been used with some success in many Ecuadorian and Mexican shrimp hatcheries (Austin et al. 1995; Vandenberghe et al. 1999; Verschuere et al. 2000). With some trials, enhancement of the larval survival, growth of shrimp and fish were clearly demonstrated compared with control groups (Maeda 1994; Austin et al. 1995). As

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well, this species displayed promise as a probiont in other species where a decrease in mortality was observed in Atlantic salmon juveniles that were bathed in V. algin-lyticus prior to pathogen challenges with Aeromonas sal-monicida, Vibrio anguillarum and Vibrio ordalii (Austin et al. 1995). Similarly, mortality of Artemia naupli chal-lenged with V. parahaemolyticus was diminished when V. alginoyticus was added to the culture water (Gomez-Gil et al. 1998). Although some strains of V. alginolyticus have been reported as pathogens (Anguiano-Beltran et al. 1998; Chen et al. 1999; Zanetti et al. 1999), this spe-cies contains numerous strains with distinctive biologi-cal properties and only a small proportion are known to be virulent (Sugumar et al. 1998; Verner-Jeffreys et al. 2003). One of the cultured seawater isolate V. algin-lyticus C7b was beta hemolytic and therefore not con-sidered a probiotic candidate (Gomez-Gil et al. 2002). Considering the potential pathogenic nature of Vibrio shrimp aquaculture, probiotic strains need to be moni-tored to reduce the possible risk of transferring into pathogenic form.

Pseudomonas

Members of the genus Pseudomonas are common inhabitants of soil, freshwater and marine environ-ments and are known to produce a wide range of sec-ondary metabolites (Raaijmakers et al. 1997) inhibit-ing a wide range of pathogenic bacteria. Vijayan et al. (2006) reported a promising antagonistic bacterium Pseudomonas PS-102, isolated from a brackish water lagoon, which showed antagonistic property towards a wide range of pathogenic vibrios isolated from penaeid and Macrobrachium larval rearing systems. Growth as well as production of the antagonistic component over a wide range of temperatures, pH and salinities suggests that the isolate Pseudomonas PS-102 could be a suitable candidate probiotic for both penaeid and non-penaeid systems. The fluorescent pseudomonads have been used as biocontrol agents in several rhizo-sphere studies where their inhibitory activity has been attributed to a number of factors, such as the produc-tion of antibiotics, hydrogen cyanide, or iron-chelating siderophores (Raaijmakers et al. 1997). Pseudomonas spp. and vibrios are the most common genera associ-ated with crustaceans (Moriarty 1998) and are com-mon inhabitants of the aquatic environment including shrimp culture ponds (Otta et al. 1999). Torrento and Torres (1996) reported the in vitro inhibition of V. har-veyi by a Pseudomonas species isolated from the aquatic environment. A marine bacterial strain, Pseudomonas I-2, produced inhibitory compounds against shrimp pathogenic vibrios including Vibrio harveyi, V. flu-vialis, V. parahaemolyticus, V. damsela, and V.vulnificus (Chythanya et al. 2002). The inhibitory substance was

found to be a low molecular weight compound, heat stable, soluble in chloroform and resistant to proteo-lytic enzymes.

Roseobacter

In an attempt to identify whether high level of larval mor-tality (“larval crash”) are indeed caused by opportunistic bacteria, Griffiths et al. (2001) monitored the succession of bacterial species associated with the development of haddock larvae between hatching and weaning in a New Brunswick hatchery. In their survey, they identified a vir-ulent pathogen of haddock (Psuedoalteromonas sp.) and also a bacterial species associated with enhanced sur-vival (Roseobacter sp.). From this, they summarized that it may be possible to improve the survival of the larvae during this vulnerable phase by preemptively colonizing the larval surfaces with selected heterotrophic bacteria. In a survey of bacterial communities associated with the early stages of Great Scallop (Pecten meximus), Sandaa et al. (2003) concluded that a significant portion of bac-teria to rearing tanks is contributed from algal culture used as feed. Their algal cultures contained 73% alpha proteobacteria, particularly those from the Rhodobacter and Roseobacter groups which are known to degrade dimethylsulfonionpropionate (DMSP), an osmoprotect-ant synthesized and stored by marine algae (Keller at al. 1989). Roseobacter clade has been reported as one of the major bacterial endosymbionts of marine invertebrates (Allgaier et al. 2003). Literature evidenced that the attempts / studies on the possible use of Rhodobacter as shrimp probiotics are scanty.

Arthrobacter

Microorganisms from the genus Arthrobacter possess many advantageous properties and nutritional benefits to be used as probiotics in aquaculture. There were many reports of Arthrobacter sp. producing antimicro-bial compounds (Hentschel et al. 2001; Jayanth et al. 2001). In addition, Arthrobacter sp. can utilize a wide and diverse range of organic substances as carbon and energy sources including nicotine, nucleic acids and various herbicides and pesticides (Keddie et al. 1986; Hagedom and Holt 1975), which in turn having biore-mediation potential. Arthrobacter sp. was also found to remove Mn2+, Cu2+, Ni2+, and Pb2+ from synthetic solu-tions (Veglio et al. 1996). As reviewed by Abrashev et al. (1998), some Arthrobacter species have the ability to produce a number of valuable substances includ-ing aminoacids, vitamins, enzymes, specific growth factors, and polysaccharides. Until now, however, no Arthrobacter sp. has been evaluated as a possible pro-biotic bacterium in aquaculture. Recently, Arthrobacter sp. designated XE-7 was used against three target strains,

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including Vibrio parahaemolyticus, Vibrio anguillarum and Vibrio nereis, and was tested for the protection of P. chinensis postlarvae from pathogenic vibrios (Li et al. 2006).

Microalgae

Microalgae based approach commonly used in aquacul-ture because of its beneficial effects to the target-organ-isms is the so-called “green-water technique” (Cremen et al. 2007). This technique is based on the addition of microalgae in closed water systems in the most critical times, when larvae are fragile, sensitive to environmen-tal changes and easily stressed (Papandroulakis et al. 2001). Numerous studies have shown enhanced growth, survival and health status of marine larvae cultured with microalgae (e.g., Isochrysis galbana, Tetraselmis suecica, Phaedactylum tricornutum, Dunaliella salina or Dunaliella tertiolecta) (e.g., Nass et al. 1992; Reitan et al. 1997; Cahu et al. 1998; Suppamattaya et al. 2005). Preetha et al. (2007) established marine cyanobacterium Synechocystis MCCB 114 and 115 as putative probionts for shrimp P. monodon. Different hypotheses have been suggested to explain the beneficial effects of algae to the larvae, such as (1) direct supply of nutrients (Reitan et al. 1997); (2) stimulation of the digestive abilities of larvae (Cahu et al. 1998); (3) influence on the bacterial population of the rearing water and thus contributing to the establishment of an early gut microbial flora in the larvae (Bergh et al. 1994; Skjermo and Vadstein 1999).

Most common strains in shrimp aquaculture

Bacteria that have been used successfully as probiotics belong to the genus Vibrio (Griffith 1995; Garriques and Arevalo 1995), Bacillus spp. (Moriarty 1998; Rengpipat et al. 1998) and Thalassobacter utilis (Maeda and Liao 1992). Most researchers have isolated these probiotic strains from shrimp culture water (Nogami and Maeda 1992; Direkbusarakom et al. 1997; Tanasomwang et al. 1998), or from the intestine of different penaeid spe-cies (Rengpipat et al. 2000). Gomez-Gil et al. (1998) demonstrated the existence of a wide diversity of Vibrio species in the hepatopancreas of healthy Penaeus van-namei. Probiotics can improve digestive activity by the synthesis of vitamins, cofactors or by improving the enzymatic activity (Fuller 1989; Gatesoupe 1999; Jory 1998; Ziemer and Gibson 1998). These properties could facilitate the weight increase, improve digestion or nutrient absorption. Though the probiotics might have potential nutritional improvement capacity, the main objective of probiotics would be to exploit their benefits by restricting the appearance of pathogenic bacteria in shrimp culture systems (Gullian et al. 2004). Most of the available reports on application of probiotics in

shrimp aquaculture are confined to control of bacterial pathogens and improvement of shrimp health. Reports on the control or management of viral pathogens are scanty. Albeit, in principal, direct effect of probiotics on viral pathogens cannot be feasible, the immune effect of probiotics particularly -1,3/1,6-glucans in Penaeus vannamei juveniles elicited protection against white spot syndrome virus challenge (Rodríguez et al. 2007). Although the exact mechanism by which these bacte-ria do this is not known, laboratory tests indicate that the inactivation of viruses can occur by chemical and biological substances, such as extracts from marine algae and extracellular agents of bacteria. It has been reported that strains of Pseudomonas sp., Vibrios sp., Aeromonas sp., and groups of coryneforms isolated from salmonid hatcheries, showed antiviral activity against infectious hematopoietic necrosis virus (IHNV) with more than 50% plaque reduction (Kamei et al. 1988). Girones et al. (1989) reported that a marine bacterium, tentatively classified in the genus Moraxella, showed antiviral capacity, with high specificity for poliovirus. Direkbusarakom et al. (1998) isolated two strains of Vibrio spp. NICA 1030 and NICA 1031 from a black tiger shrimp hatchery. These isolates displayed antiviral activities against IHNV and Oncorhynchus masou virus (OMV), with percentages of plaque reduction between 62 and 99%, respectively.

The efficacy of probiotics in disease / health manage-ment has been well established in Litopenaeus vannamei. Four bacterial strains isolated from the gastrointestinal tract of adult shrimp L. vannamei including V. algino-lyticus UTM 102, B. subtilis UTM 126, Roseobacter gal-laeciensis SLV03, and Pseudomonas aestumarina SLV22, were showed antagonism against the shrimp-pathogenic bacterium, V. parahaemolyticus PS-017 (Balcázar et al. 2007). It has also been reported that lactic-acid bacteria increase the survival of marine shrimp, L. vannamei, after infection with Vibrio harveyi (Vieira et al. 2007). This study evaluated the survival, post-larvae quality, and the population of bacteria in L. vannamei after the addition of two strains of lactic-acid bacteria (2 and B6) experi-mentally infected by V. harveyi. The survival of control shrimp was lower (21%) than that of animals fed with the strains B6 (50%) and 2 (44%). Albeit the study showed the efficacy of lactic acid bacteria on the control of V. harveyi, the efficacy cannot be considered as significant, since the survival of treated shrimp was 29% and 23% respectively over the control. Balcázar and Rojas-Luna (2007) demonstrated inhibitory activity of probiotic B. subtilis UTM 126 against vibrio species confers protec-tion against vibriosis in juvenile L. vannamei. Li et al. (2007) reported beneficial effects of Bacillus licheniformis on the intestinal microbiota and immunity of the white shrimp, Litopenaeus vannamei. The administration of B. licheniformis improved the white shrimp’s intestinal

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microbiota, and its immune ability. Supplementation of prebiotic compounds, including short-chain fructoo-ligosaccharides has been shown to confer benefits on nutrient utilization, growth, and disease resistance of L. vannamei through improved gastrointestinal microbiota (Li et al. 2007a). Thus, the application of probiotics gets effective on the stabilization and improvement of gut microbiota in shrimp. Though shrimp immune system and physiology are different from higher animals, one of well-established mechanisms of action of probiotics in higher animals, stabilization of gut microbiota appear to be functional in shrimp body. Recent studies evidenced that shrimp immune system could be enhanced with the administration of probiotics. Chiu et al. (2007) demon-strated that administration of Lactobacillus plantarum in the diet induced immune modulation and enhanced the immune ability of L. vannamei, and increased its resistance to V. alginolyticus infection. However, the total hemocyte counts significantly decreased in shrimp fed with diet containing Lac. plantarum. Activities of pro-tease and amylase, two digestive enzymes of the mid-gut gland and the intestine were significantly (P < 0.05) higher in Bacillus probiotic-treated L. vannamei shrimp than in the control (Gómez and Shen 2008).

Possible mode of action

ImmunomodulaionThe functional role of yeasts in the gut particularly its occurrence, dietary effects and fish health development has been reported recently (Gatesoupe 2007). In fish, it has been demonstrated that probiotic supplementation enhances immune function, such as phagocytosis by neutrophils and macrophages isolated from the head kidney (Villamil et al. 2002; Panigrahi et al. 2004; Balcazar et al. 2007). Moreover, recent studies have addressed the effect of probiotics on mucosa-associated lymphoid tissue in fish (Balcázar et al. 2006; Picchietti et al. 2007 2008; Gómez and Balcázar 2008). Epithelial cells that line the intestinal tract are known to take part in initiation and regulation of the mucosal immunity to bacteria by interacting with underlying immune cells, such as, mac-rophage, neutrophils, eosinophils, natural killer cells, and intraepithelial lymphocytes (Kagnoof and Eckmann 1997). The importance of ‘cross-talk’ between epithelial cells and gut bacteria and cytokine signals between epi-thelium and immune cells has been highlighted (Delneste et al. 1998). Recently, Kim and Austin (2006) reported the impact of probiotics on cytokine mRNA expression in head kidney leucocytes and gut cells isolated from rainbow trout. Recent studies have also demonstrated the ability of isolated gut leukocytes to perform phago-cytosis and secrete bactericidal oxygen free radicals (Davidson et al. 1991; Clerton et al. 1998). Phagocytosis is a form of endocytosis, mediated by phagocytic cells

such as neutrophils, monocytes and macrophages, where large particles (i.e. microorganisms) are ingested into endocytic vesicles called phagosomes. The funda-mental role of these cells in host defense is to limit the initial dissemination and /or growth of infectious organ-isms (Neumann et al. 2001). Few studies even described the acquisition of long lasting immune states following exposure of prawns to killed Vibrio sp. due to humoral factors, cellular factors and antibacterial activity (Itami et al. 1989; Alabi et al. 1999 2000). The immunomodulatory potential of probiotics are well studied in fish (Picchietti et al. 2007; Panigrahi et al. 2007) but studies in the shrimp is restricted to few strains.

Competitive exclusionCompetitive exclusion is one of the ecological proc-esses that can be manipulated to modify the species composition of a soil or water body or other micro-bial environment. Small changes in factors that affect growth or mortality rates will lead to changes in species dominance. We are still a long way from knowing all the factors that control bacterial growth rates and even the complete species composition by making use of the competitive exclusion principles (Smith and Davey 1993). Competitive exclusion based on the nutritional selectivity may not be adequate for potential shrimp probiotics. The potential strains having non-specific antagonistic property and broad spectrum of nutritional niche including gastrointestinal tract and detritus might be the most promising strains for shrimp aquaculture (Farzanfar 2006).

BioremediationMicrobial ecology and biotechnologies have advanced in the last decade, to the point that commercial products and technologies are available for treating large areas of water and land to enhance population densities of par-ticular microbial species and/or targeted activities. The practice of bioremediation or bioaugmentation is applied in many areas, but success varies greatly, depending on the nature of the products used and the technical infor-mation available to the end user. The bacteria that are added must be selected for specific functions that are amenable to bioremediation (Lin 1995; Rengpipat et al. 1998), and be added at a high enough population den-sity, and under the right environmental conditions, to achieve the desired outcomes. Bioaugmentation and the use of probiotics are significant management tools, but their efficacy depends on understanding the nature of competition between species or strains of bacteria. They rely on the same concepts that are used successfully from soil bioremediation and probiotic usage in the ani-mal industry. The effects of probiotics on the sediment of P. vannamei pond during 117 days of culture period were reported by Wang et al. (2006). In this study, the

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authors claimed the probiotics application significantly decreased the concentrations of total nitrogen, total phosphorous, and sulfide in sediment, but no significant difference was observed in total plate count of microbes between treated and control ponds. The functional role of probiotics in detritus management system and sig-nificant reduction of pathogenic bacterial load in the sediment of treated ponds was well documented but the concomitant effect on the pond water and shrimp body is not available.

Source of nutrients and enzymatic contribution to digestionSeveral studies on the beneficial effects of probiotics in the digestive process of the fish have been reported. However, reports on mechanisms of probiotics in shrimp digestive process are scanty. In the present review the available reports in fishes are considered as a possible mode of action in shrimp. Bacteria have been isolated from fish intestine that produce various vitamins as secondary metabolites. Vitamin B12 is produced by different bacteria isolated from the microbiota of a variety of fish species, including carp (Cyprinus carpio) (Kashiwada et al. 1970; Sugita et al. 1990; Sugita et al. 1991), channel catfish (Ictalurus punctatus) (Limsuwan and Lovell 1981), and various tilapia species (Lovell and Limsuwan 1982; Sugita et al. 1991). Ringø et al. (1995) suggested that species of Agrobacterium, Pseudomonas, Brevibacterium, Microbacterium, and Staphylococcus isolated from the intestinal tract of alpine trout (Salvelinus alpinus L.) contributed to the nutritional process by the production of lipases that increase the absorption of lipids. In fish, it has been reported that Bacteroides and Clostridium sp. have contributed to the host’s nutrition, especially by supplying fatty acids and vitamins (Sakata 1990). It has been established that some bacteria may participate in the digestion proc-esses of bivalves by producing extracellular enzymes, such as proteases, lipases, as well as providing neces-sary growth factors (Prieur et al. 1990). Similar obser-vations have been reported for the microbial biota of adult penaeid shrimp (Penaeus chinensis), where a complement of enzymes for digestion and synthesize compounds that are assimilated by the animal (Wang et al. 2000). In addition, a study in oscar (Astronotus ocel-latus), fish angel (Pterophyllum scalare), and southern flounder (Paralichthys lethostigma) suggested that the intestinal anaerobic bacteria can play a role in the diges-tive process of the fish by providing a variety of enzymes such as cabohydrases, phosphatases, esterases, lipases, and peptidases that help in the absorption of nutrients (Ramirez and Dixon 2003). Microbiota may serve as a supplementary source of food and microbial activity in the tract digestive may be a source of vitamins or essen-tial amino acids (Dall and Moriarty 1983).

Quorum sensing blockingRecently, a new mechanism of action of shrimp gut microbiota has been demonstrated by Tinh et al. (2007). It has been established that quorum sensing blocking is one of the emerging and effective disease management strategy against Vibrio spp. in shrimp. N-acyl homoser-ine lactone mediated quorum signaling is a well-estab-lished mechanism of swarming, biofilm formation and pathogenesis in aquaculture systems. N-acyl homoser-ine lactone-degrading microbial enrichment cultures isolated from P. vannamei gut was found to have poten-tial probiotic properties in Brachionus plicatilis cultures (Tinh et al. 2007). This paper brings out a new insight on the mode of action of probiotics in shrimp aquaculture.

Probiotics in shrimp aquaculture: field realities

Although probiotics are emerging as promising venture to prevent mid-culture outbreaks in farmed shrimp, reports on the efficacy, economy, and viability of pro-biotic application in shrimp farms are scanty. Moriarty (1999) reported on his successful experiences of using probiotic bacteria instead of antibiotics to control lumi-nous vibrios in shrimp farms in Negros, Philippines. The field trials for the evaluation of efficacy of bacterial pro-biont Bacillus S11 supplemented feed (PF) was carried out in 2 earthen ponds of black tiger shrimp Penaeus monodon for 100 d during 2 different seasons (hot and cool) in Thailand (Rengpipat et al. 2003). They compared the growth and survival of shrimp with those receiving an unsupplemented feed (UF). The shrimp fed PF grew significantly larger and had significantly higher survival than shrimp fed UF (p < 0.05). They projected yields on an annual basis (two 100 d crops) were 49% greater with PF-fed shrimp. In another field study, the efficacy of a commercial microbial product was tested in tiger shrimp, Penaeus monodon (Fabricius), ponds for one culture period in Kuala Selangor, Malaysia (Shariff et al. 2001). The use of probiotics in shrimp aquaculture has been established as proven technology in Ecuadorian shrimp hatcheries and culture systems (Balcazar 2003). The use of Vibrio alginolyticus strains as a probiotics has been recommended to increase survival and growth of white shrimp (Litopenaeus vannamei) post-larvae in Ecuadorian hatcheries. Competitive exclusion of poten-tial pathogenic bacteria effectively reduces or eliminates the need for antibiotic prophylaxis in intensive larvicul-ture systems (Garriques and Arevalo 1995).

The effectiveness on water quality, population den-sity of bacteria, and shrimp production in ponds treated with commercial probiotics was tested in Penaeus van-namei ponds in Hai-yan, China (Wang et al. 2005). They found the shrimp from ponds treated with probiotics as bioremediation had a significantly higher survival rate, feed conversion rate, and final production over the

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control ponds. This indicates that the addition of the commercial probiotics had a noticeable influence on shrimp production and survival rate. According to Wang et al. (2005), the probiotics tested in the field conditions elicited the following benefits over the control ponds.

Improved pond water quality and lower rate of water •deterioration.Improved microecology of the shrimp ponds•Attainment of a dynamic balance between probiot-•ics and pathogensEnhanced the number of beneficial microbial com-•munities such as protein mineralizing bacteria, Bacillus sp., and ammonifying bacteriaEnhanced decomposition of organic mat-•ter and reduction in nitrogen and phosphorus concentrationsBetter algal growth and higher dissolved oxygen •concentration

Wang et al. (2005) also envisaged the need of fur-ther research to understand the functional mechanism among the microorganisms, and how the probiotics work in ponds. Nunes (2005) tested the efficacy of a commer-cial probiotics-based product (Efinol PT, manufactured by Bentoli, Inc. USA) in two pond trials at the commer-cial shrimp farm located in the northeastern region of Brazil. The product is claimed to be a combination of beneficial microorganisms including Bacillus subtilis, B. licheniformis, B. coagulans, Lactobacillus acidophilus, Streptococcus faecium, and Saccharomyces cerevisiae that colonize the pond water and the intestinal walls of aquatic animals. These findings provide evidence that a reduction in mortality due to Infectious Myonecrosis Virus is associ-ated with the probiotics product application. The suscep-tibility of the shrimp to infection is not completely elimi-nated by the probiotics, but there is a 2-week delay in the onset of the disease and mortality. Mathieu et al. (2008) studied the effects of a lactic acid bacterium, Pediococcus acidilactici as a dietary probiotic on growth performance and some nutritional and microbiological aspects of the shrimp Litopenaeus stylirostris. It was found that the probiotic improved production in the treated ponds with increases in the survival rate (7% and 15% respectively) and final biomass (8% and 12% respectively). Albeit, this study demonstrated, under pond conditions, that feeding shrimp with live terrestrial lactic acid bacteria can be an effective treatment for improving shrimp culture affected by vibriosis, the efficacy on survival of treated shrimp are not significantly over control and the stability of the cul-ture for long-term applications need to be explored.

The synergistic use of ozone and probiotics was found to be effective on the survival of black tiger shrimp (Penaeus monodon) (Meunpol et al. 2003). Reports evidenced the synergistic formulations are highly effec-tive in the field condition than microbial additives. For

example, the shrimp survival after probiotic treatment, coupled with ozonation, increased significantly com-pared with controls (Meunpol et al. 2003). In principle, bacteria added directly to pond water are not probiotics, and should not be compared with living microorganisms added to feed (Rengpipat et al. 2003). Many workers have evaluated some specific microorganisms as biological improvers for water quality: Douilett (1998) developed a probiotic blend to improve water quality in fish and crustacean cultures by reducing the concentration of organic metabolites and ammonia. The addition of this blend to culture systems reduced the concentration of Vibrio strains and thus controlled diseases caused by Vibrio strains. In shrimp ponds, the mineralization of organic matter can be accelerated by the application of probiotic bacteria. The application of commercial micro-bial products in aquaculture ponds is rapidly increasing as a proactive disease management strategy (Gatesoupe 1999). However, this mineralization process depends on the density of suitable microbiota, rate of substrate incorporation, and suitable environmental conditions (Rodina 1972). Under poor bottom conditions, even commercial bacterial products cannot carry out the mineralization and degradation efficiently (Shariff et al. 2001). Based on the available reports of field level effi-cacy of probiotics on shrimp production, such products cannot be guaranteed a substantial improvement in the water quality and shrimp production in different shrimp farms. Since the field-level efficacy is greatly depend on stocking density, shrimp species, quality of feed, level of water exchange, history of shrimp farm under probiotic treatment, combination of probiotic strains, inlet water quality, etiological factors and regional climatic factors. Therefore, a particular probiotic product cannot be gen-eralized for all type of shrimp farms in different regions. Further, the cost-benefit analyses of probiotic products in field-level trails are inevitable to commercialize such products.

Shrimp probiotics: can be a sustainable venture?

Though many available reports validate the potential of probiotics in shrimp aquaculture, the shelf life of pro-biotics and sustainability of such products in the entire culture cycle and/or transform to be a native strain (autochthonous) for a longer duration is not known. Homeostasis of bacterial communities is represented by a steady state that is generated by the organisms them-selves. Competition for nutrients and space, the inhibi-tion of one group by the metabolic products of another group, and predation and parasitism all contribute to the regulation of populations in particular proportions, one to the other. Based on the homeostasis phenom-enon (Alexander 1971), the allochthonous microbiota seems to have a seldom chance to be an autochthounous

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microbiota. Because all of the ecological niches are filled in a regulated bacterial community, it is extremely difficult for allochthonous (formed in another place) microbes, accidentally or intentionally introduced into an ecosystem, to establish themselves (Tannock 2004). Therefore, sustainability of probiotics in the shrimp farms is a complex ecological phenomenon, require a multidimensional approach to find out the prebiotic factors essential for the establishment and propagation of allochthonous probiotic microorganism in shrimp farm. Alvandi et al. (2004) determined Pseudomonas sp. PM11 and Vibrio fluvialis PM17 isolated from the gut of farm-reared subadult shrimp as candidate probionts and tested for their effect on the immune factors of black tiger shrimp. The results of the study suggest that the criteria used for the selection of putative probiotic strains, such as predominant growth on primary isolation media, abil-ity to produce extracellular enzymes and siderophores, did not bring about the desired effect in vivo and improve the immune system in shrimp. Though the lyophilized cultures are proven to be successful in human (Hong et al. 2005) and veterinary applications, their efficacy in aquaculture systems are being an unresolved enigma.

There are no serious changes during the initial stages of farming aquatic organisms, when the stocked biomass are small and their metabolism rate and amounts of sup-plementary feed are low. However, towards the progress of culture leading to a rapid increase in biomass, con-sequently water quality deteriorates, mainly as a result of the accumulation of metabolic waste of cultured organisms, decomposition of unutilized feed, and decay of biotic materials (Prabhu et al. 1999). Thus, the mid-culture outbreaks in the shrimp farms are highly devas-tative. Therefore the application of probiotics needs to be rationalized and optimized for the reduction of toxic metabolites / bacterial load during late mid-culture period, instead of applying probiotics as feed additive or water enrichments for the entire culture cycle, which ultimately increase the cost of production.

It was found that production of crab (Portunus trituberculatus) larvae increased following the addi-tion of bacterial strain PM-4 daily to the culture water (Nogami and Maeda, 1992). If probiotics are to be persisted to make any sustained contribution to the indigenous microbiota, their introduction would need to be on a regular basis and/or at a concentration higher than that of the already established microbial community (Verschuere et al. 2000). The dose-effect relationship must be carefully determined so as to avoid overdosing, which may result in lower efficacy while increasing costs, or conversely, underdosing, which reduces the efficacy of the probiont (Vine et al. 2006). By their sustained presence, periodic additions may also assist in maintaining the desired microbiota at levels that inhibit the development of pathogenic

or opportunistic bacteria. It has been suggested that aquaculture systems cannot support bacterial con-centrations greater than 106 cells mL−1 (Maeda 1994). Therefore, more information needed on the selection of suitable strains in the correct numbers for use at an appropriate schedule for improving the pond condi-tion and yield.

Conclusion

It has been envisaged that cost-effective holistic/inte-grated management system is inevitable for sustainable shrimp farming. The existing proactive management strategies using immunostimulants / natural prod-ucts can only ensure shrimp health to certain extent but none of the product(s) ensure the management of shrimp and environment (detritus) holistically. As such probiotics are the only emerging / promising proac-tive strategy which could ensure both shrimp health and environmental management holistically. Though the probiotics in shrimp health management has been well documented, its bioremediation potential and biocontrol (antagonism) strategies need to be resolved. In order to obtain sustained probiotic effect in the field conditions, it is obligatory to screen the vast marine microbes instead of utilizing terrestrial strains. In this regard, the marine bacterial endosymbionts are emerg-ing as a potential resource for developing cost-effective safe probiotics for shrimp aquaculture.

Acknowledgements

JS thankfully acknowledge the research grant by University Grants Commission (UGC), New Delhi on the development of “Shrimp probiotics from marine bacterial symbionts”.

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probiotics in shrimp aquaculture: avenues and challenges

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