Probiotics in Aquaculture: A Close Look

This article provides an overview on probiotics in aquaculture.

Introduction:

The concept of probiotics is not as recent as many think.

The earliest published comments on probiotics relate to observations from the early 20th century by the Nobel winning scientist, E. Metchnikoff (1907, 1908 ; cited by Tannock, 1997) who suggested that the long life of Bulgarian peasants resulted from the consumption of fermented milk products containing bacteria, which positively influenced the flora of their colon.

As the concept evolved, many products primarily in the agricultural and human health related areas have developed.

The scientific basis of the concept is sound, though somewhat controversial. The modern concept of probiotics was formulated only about 30 years ago (Parker, 1974).

However, its pertinence was challenged for many years among the scientific community. The history of the probiotic effect has been documented many times previously (Bibel, -1982; Fuller, 1992). Long before their discovery, microbes have been unawarely used to preserve food and these empirical methods contributed to improve human health (Bengmark, 1998).

Disease outbreaks are recognized as one of the significant constraints on aquaculture production and trade, affecting the economic development of the sector in many countries. So far, conventional approaches, such as the use of disinfectants and antimicrobial drugs, have had limited success in the prevention or cure of aquatic diseases.

Moreover, there is a growing concern about the use and, particularly abuse, of anti-microbial drugs, not only in human medicine and agriculture but also in aquaculture. The emphasis in disease management should be on prevention, which is likely to be cost-effective than cure.

This may lead to less reliance on the use of chemicals, such as antimicrobials, disinfectants, and pesticides, which largely treat the symptoms of the problem and not the cause (Planas and Cunha, 1999). According to Browdy (1998), one of the most significant technologies that have evolved in response to disease control problems is the use of probiotics.

Considering the recent successes of these alternative approaches, the Food and Agricultural Organization of the United Nations (Subasinghe, 1997) defined the development of affordable, yet efficient vaccines, the use of immunostimulants, and nonspecific immune enhancers, and the use of probiotics and bio-augmentation for the improvement of aquatic environmental quality as major areas of further research in disease control in aquaculture. The results of this research will undoubtedly help to reduce the use of chemical and drug in aquaculture and will make aquaculture products more safe and acceptable to consumers.

The aim of the present review is to provide an overview of the work done on the role of probiotic bacteria in aquaculture with special reference to their application as biological control agents for aquaculture environments. In addition, the role of probiotics in fish nutrition has also been discussed.

What are Probiotics?

A number of definitions of probiotics have been successively proposed. The term probiotics was first used by Lilly and Stillwell (1965) who proposed the definition “substances produced by one protozoan that stimulated the growth of another”. Later, Parker (1974) defined probiotics as “organisms and substances which contribute to intestinal microbial balance”. However, this definition included antibiotics and short-chained fatty acids also.

The definition was then restricted to “a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance” (Fuller, 1989). It has been stated that the interaction between the microbiota, including probiotics, and the host is not limited to the intestinal tract. Probiotic bacteria could also be active on the gills or the skin of the host as well as in its ambient environment.

The intensive interaction between the culture environment and the host in aquaculture implies that a lot of probiotics are obtained from the culture environment and not directly from feed, as stipulated by the definition of Fuller (1989). Tannock (1997) noted that the effect on the “intestinal microbial balance” has not been demonstrated in most cases, and he further proposed the definition of probiotics as “live microbial cells administered as dietary supplements with the aim of improving health”.

Moriarty (1998) wanted to extend the definition of probiotics to “microbial water additives”. Gatesoupe (1999) suggested an alternative definition of probiotics as “microbial cells that are administered in such a way as to enter the gastrointestinal tract and to be kept alive, with the aim of improving health”.

Recently, Verschuere et al. (2000) proposed a modified definition which allows a broader application of the term “probiotic” and addresses to the objections made earlier. According to them, 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.

Based on this definition, probiotics may include microbial adjuncts that prevent pathogens from proliferating in the intestinal tract, on the superficial structures, and in the culture environment of the cultured species, that secure optimal use of the feed by aiding in its digestion, that improve water quality, or that stimulate the immune system of the host. Bacteria delivering essential nutrition to the host (single-cell protein) without being active in the host or without interacting with other bacteria, with the environment of the host, or with the host itself are not included in this definition.

How do Probiotics Act?

The use of probiotics has long traditions in animal husbandry (Stavric and Kornegay, 1995), but is rarely applied in aquaculture. In spite of extensive efforts destined for treatment of various conditions in different animals, little is known of the way in which probiotics act. Misra (1997) summarized some possible ways by which a probiotic may exert its beneficial effects.

These are:

a. By modifying metabolic processes of the host.

b. By suppressing reactions that cause generation of toxic metabolites.

c. By stimulating enzyme reactions involved in detoxification of toxic substances.

d. By stimulating activity of enzymes involved in digestion of complex nutrients or supplementing the enzymes by bacterial source.

e. By synthesizing vitamins and other essential nutrients not provided in sufficient quantities in the diet.

However, there are likely to be some other mechanisms also to operate the effects of probiotics in different forms.

Selection Criteria for Probiotics:

The initial major purpose of using probiotics is to maintain or establish a favourable relationship between friendly and pathogenic microorganisms that constitute the flora of intestinal or skin mucous of fish. A successful probiotic is expected to have a few specific properties in order to certify a beneficial effect. Antagonism to pathogens is one such property of a probiotic bacterium.

One sign of antagonism is that it produces antimicrobial substances like organic acids, hydrogen peroxide, or siderophores. In order to have a beneficial effect in the form of growth promotion or to protect fish against bacterial pathogens, the strains should also have the capacity to colonize the fish by adhesion, and to produce important substances, like vitamins.

The microorganisms should be viable for long periods under storage as well as field conditions. Adhesion is one of the most important selection criteria for probiotic bacteria, because it is considered as a prerequisite for colonization. Probiotic bacteria will, of course, have to be non-pathogenic, non-toxic in order to avoid undesirable side effects when administered to fish. Test of antagonism, studies of adhesion, and challenge tests in vitro are essential to select the probiotic candidates. Challenge experiments where fish treated with friendly bacteria are subjected to pathogens are also needed.

Different Forms of Probiotics:

The lack of fundamental knowledge about the mechanism of the probiotic effect has not deterred the development of probiotic preparations. The preparations vary in the way in which they are presented. They may be in form of powders, tablets, pastes or sprays with different excipients to maintain the preparation in the required condition (Fuller, 1997).

There are a variety of such probiotic products available in the market that claim to have many effects including improved resistance, antitumour activity, increased growth rate and feed conversion in farmed animals, improved milk production by cows or increased egg production by poultry birds.

Application of Probiotics in Aquaculture:

The theory of ecological prevention and cure in controlling the insect pest of terrestrial domesticated animals and plants has been a practice for long time and has achieved remarkable success. The use of beneficial bacteria in human and animal nutrition is well documented. Only in the late 1980s did the first publications on biological control in aquaculture emerge, and since then the research effort has continually increased.

Generally, probiotics are applied in the feed or added to the culture tank or pond as preventive agents against infection by pathogenic bacteria, although nutritional effects are also often attributed to probiotics, especially for filter feeders (Verschuere et al. 2000).

Most probiotics used as biological control agents in aquaculture belong to the lactic acid bacteria (Lactobacillus, Caranobacterium, etc.), to the genus Vibrio (Vibrio alginolyticus, etc.), Bacillus, or to Pseudomonas, although other genera or species have also been mentioned. An overview of the published reports dealing with probiotics as biological control agents in aquaculture is given in Table 1.

The first trials of incorporation of probiotics into aquaculture feeds used commercial preparations designed for land animals. Spores of Bacillus toyoi isolated from soil reduced mortality of Japanese eel which were infected with Edwardsiella sp. (Kozasa, 1986). The same strain increased the growth rate of yellowtail (Kozasa, 1986) and when tested on rotifers (Brachionus plicatilis), the treatment increased the growth rate of larval turbot (Gatesoupe, 1989).

Recently, the bio-controlling theory has been applied to aquaculture. Attempts have been made by many researchers to use some kind of probiotics in aquaculture water to regulate the micro-flora of aquaculture water, control pathogenic microorganisms, to enhance decomposition of the undesirable organic substances in aquaculture water, and improve the ecological condition of aquaculture. Maeda and Nogami (1989) reported some aspects of bio-controlling method in aquaculture.

In their study, they used bacterial strains possessing vibrio static activity which improved the growth of prawn and crab larvae. By applying these bacteria in aquaculture, a biological equilibrium between competing beneficial and harmful microorganisms was produced. The results indicated that the population of Vibrio spp., which frequently causes large scale damage to the larval production, was decreased. Survival rate of the crustacean larvae in these experiments was much higher than those without the addition of bacterial strains.

The results of this study suggest that controlling the aquaculture ecosystem using bacteria and protozoa is quite possible and, if this system is adopted, it will maintain the aquaculture environment in better condition, which, in turn, will increase the production of fish and crustaceans.

Porubcan reported bacterial treatments to improve production of Penaeus monodon. Floating biofilters with nitrifying bacteria decreased the amount of ammonia and nitrate in the rearing water and increased shrimp survival. The introduction of Bacillus spp. in proximity to pond aerators reduced COD and increased shrimp harvest . Nogami and Maeda (1992) isolated a bacterial strain from a crustacean culture pond.

The bacterial strain was found to improve the growth of crab (Portunus trituberculatus) larvae and repress the growth of other pathogenic bacteria, especially Vibrio spp., but did not kill or inhibit useful micro algae in sea water when it was added into the culture water. Among the bacterial population present in the culture water of the crab larvae, the numbers of Vibrio spp. and pigment bacteria decreased or even became undetectable when the bacterium was added into culture water.

The production and survival rate of crab larvae were greatly increased by addition of the probiotic bacteria into the culture water. They suggested that the bacterium might have improved the physiological condition of the crab larvae by serving as a nutrient source during its growth.

Austin et al. (1992) reported the use of a kind of micro algae (Tetraselmis suecica), which is able to inhibit pathogenic bacteria of fish. When used as a food supplement, T. suecica was found to inhibit Aeromonas hydrophila, A. salmonicida, Serrsita liquefaciens, Vibrio anguillaram, V. salmonicida and Yersinia ruckeri type I. They suggested that there may be some bioactive compounds in the algal cells, and there appears to be a significant role for Tetraselmis in the control of fish diseases.

Subsequently, Austin et al. (1995) reported a probiotic strain of Vibrio alginolyticus which did not cause any harmful effect in salmonids. When the freez-dried culture supernatant was added to the pathogenic bacteria such as, V. ordalii, V. anguillarum, A. salmonicida and Y. ruckeri, a rapid or steady decline in the number of culturable cells, compared to the controls was observed.

Their results indicated that application of the probiont to Atlantic salmon culture led to reduction in mortalities when challenged with A. salmonicida and to a lesser extent V. anguillarum and V. ordalii. Garriques and Arevalo (1995) reported that the use of V. alginolyticus as a probiotic agent may increase survival and growth in Penaeus vannamei post larvae by competitively excluding pathogenic bacteria, and can effectively reduce or eliminate the need for antibiotic prophylaxis in intensive larvae culture system.

In their study, the addition of the bacteria V. alginolyticus as a probiotic to mass larvae culture tanks resulted in increased survival rates and growth over the controls and the antibiotic prophylaxes. All these studies with this probiotic Vibrio are encouraging, and it appears that there is tremendous potential for the use of such probiotics in aquaculture as part of disease control strategy.

Maeda and Liao (1992) reported on the effect of bacterial strains obtained from soil extracts on the growth of larvae of tiger prawn, Penaeus monodon. Higher survival and molt rates of prawn larvae were observed in the experiment treated with soil extract, and the bacterial strain which promoted the growth of prawn larvae was isolated. Douillet and Langdon (1994) have reported the use of probiotics for the culture of larvae of the Pacific oyster, Crassostrea gigas.

They added probiotic bacteria as a food supplement to xenic larval cultures of the oyster which consistently enhanced growth of larvae during different seasons of the year. They suggested that the mechanism of action of probiotic bacteria are providing essential nutrients that are not present in the algal diets or improving the oyster’s digestion by supplying digestive enzymes to the larvae or removing metabolic substances released by bivalves or algae.

Qiao Zhenguo et al. (1992) studied the effect of photosynthetic bacteria in the diet of the prawn, Penaeus chinensis. Addition of the photosynthetic bacteria in the food or culture water was found to improve the growth of the prawn and the quality of the water. Cui Jingjin et al. (1997) have reported on the application of photosynthetic bacteria in the hatchery rearing of P. chinensis.

They used a mixture of several kinds of photosynthetic bacteria (Rhodomonas sp.) as water cleaner and auxiliary food. Their results showed that the water quality of the pond treated with the bacteria was remarkably improved, the fouling on the shell of the larvae was reduced, the metamorphosis time of the larvae was one day or even earlier, and the production of post-larvae was more than that of the control. Jiravanichpaisal et al. (1997) reported the use of Lactobacillus sp. as the probiotic bacteria in the giant tiger shrimp, P. monodon.

They designed an experiment to investigate an effective treatment of Lactobacillus sp. against vibriosis and white spot diseases in P. monodon. Queiroz and Boyd (1998) confirmed that a commercial inoculum of Bacillus sp. increased survival and production of channel catfish. Kennedy et al. (1998) isolated a strain of B. subtilis from the common snook, Centropomus undecimalis and inoculated into the rearing water.

The treatment resulted in the apparent elimination of Vibrio sp. from the larvae of snook after decreasing salinity level. Moriarty (1998) also documented an increased survival of prawn in ponds where some strains of Bacillus spp. were introduced. However, the effect on prawn survival might be due to either to a probiotic effect or to some indirect effect on animal health.

Commercial preparations with live lactic acid bacteria when introduced into the medium of live food organisms for larval flatfish, some of the treatment increased the production of rotifers and growth of turbot and Japanese flounder (Gatesoupe, 1989, 1991; Gatesoupe et al. 1989). Caranobacterium divergens isolated from Atlantic cod improved survival of Atlantic cod fry. In another study, commercial preparations of Streptococcus faecium improved the growth and feed efficiency of Israeli carp.

Although attempts have been made to study the probiotic effects of both commercial and autochthonous strains of microbes in aquaculture feeds by several workers abroad, very few reports are available on the use of probiotics in formulation of aquafeeds for indigenous culturable fish. Mohanty et al. (1993, 1996) reported the beneficial effects of a commercial probiotic, ‘Bioboost Forte’, in larval rearing of Indian major carps, Labeo rohita and Catla catla.

However, very little information is available concerning the bacterial population in the gastrointestinal tract of Indian freshwater fish and their role in nutrition of the host. Most attempts, therefore, have been aimed at seeking autochthonous strains with probiotic properties.

Can Microbial Communities be Manipulated?

Aquaculture practices do not provide appropriate environments for the establishment of stable microbial communities because of discontinuous culture cycles, disinfecting or cleaning of ponds or tanks prior to stocking, and sudden increase in nutrients due to exogenous feeding. Microbial communities are influenced by deterministic and stochastic factors. Deterministic factors include salinity, temperature, oxygen concentration, and quantity and quality of feed. Chances favour organisms which happen to be in the right place at the right time to enter the habitat and to proliferate if conditions are suitable.

Instead of allowing spontaneous primary colonization of the rearing water by bacteria accidentally present, the water could be preemptively colonized by the addition of probiotic bacteria. It is recognized that preemptive colonization extends the reign of pioneer organisms. A single addition would suffice if the probiotic bacteria are well adapted to the prevailing environmental conditions.

If the host or the environment already carries a stable microbial community, repeated inoculations will be necessary. It is accepted that fish contain a specific intestinal micro-biota that is established at the juvenile stage. It is unlikely that a single inoculum of probiotic bacteria will result in long term dominant colonization.

It will be necessary to supply the probiotic on a regular basis if a continuous colonization at high densities is required, especially if the bacterial species used do not belong to the normal dominant intestinal micro-biota of the cultured species or its particular development stage.

Why Autochthonous Microbes?

The commercial probiotics used for farmed animals played an important role to grow the concerns about bacterial additives in aqua-feeds. However, questions arise regarding their survival in the gastrointestinal tract of aquatic animals.

An Effective Probiotic should have some Specific Characteristics, such as:

a. It should have the ability to survive in the intestine.

b. It must produce a beneficial effect in the host animal.

To avoid the contradiction, search for autochthonous strains with probiotic properties have been initiated. The main strategy is to isolate intestinal bacteria with favorable properties from mature animals and include them in the feed for immature animals of the same species. Sugita and Shibuya (1996) reported the antibacterial abilities of intestinal bacteria in freshwater fish culture.

They isolated bacteria from the intestine of seven species of freshwater cultural fish, and investigated the antibacterial abilities of these bacteria to eighteen fish or human common pathogenic bacteria. The results of their study indicated that bacteria isolated from the intestine of fish possess the antibacterial abilities, and the presence of the intestinal bacteria can protect the fish against the infection by pathogenic bacteria. The use of an organism normally found in symbiotic association with the host will better ensure its survival and beneficial effects for the host.

Conclusion:

The use of probiotic bacteria has tremendous scope and the study of the application of probiotics in aquaculture has a glorious future. Research should be focused to find out the influence of diet on the gut micro-flora and the influence of probiotics on the existing gastrointestinal micro-flora of aquatic animals.

There is a great need to ensure the fate of probiotics in the gastrointestinal tract of the host and technical solutions to keep the probiotics alive in dry pellets. Age is an important factor for growth, and may be for probiotic response also. The same probiotic strain may respond in different way at different stages of life.

Further research and development in this field may emerge out the best way for the introduction of probiotics in aqua-feeds to explore their full potential. Probiotics principally inhibit growth and decrease pathogenicity of the pathogenic bacteria, improve the nutrition of aqua-cultured animals, improve the quality of the aquaculture water. Probiotics also decrease the use of antibiotics and other chemicals, thereby decreasing environmental contamination by the residual antibiotics and chemicals. The benefit of using probiotics will be long lasting and the application of probiotics will become a major field in the development of environmental-friendly aquaculture in the future.