The digestive tract is one of the common sites where pathogens could enter into the shrimp; colonization by microbiota of mucus in the gastrointestinal tract acts as the first line of defense against pathogenic, exogenous or opportunist microorganisms, which establishes a barrier effect. A group of non-digestible food ingredients which could selectively stimulate the growth and/or the metabolism of health-promoting bacteria in the intestinal tract, and thus improve an organism’s intestinal balance, is known as prebiotics.
This concept of “prebiotic” was introduced by Gibson and Roberfroid in 1995. One decade later, the refined prebiotics definition was given by Roberfroid as follows: “A prebiotic is a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health.” Three criteria were further stated for dietary carbohydrates to be considered as prebiotics: 1) resistance to gastric acidity, to hydrolysis by mammalian enzymes, and to gastrointestinal absorption; 2) fermentation by intestinal microflora; and 3) selective stimulation of the growth and/or activity of those intestinal bacteria that contribute to health and well-being.
Beneficial effects of prebiotics were first acknowledged in the terrestrial mammals, and primarily as humans’ dietary supplements. The application of prebiotics has also attracted increased interest in aquaculture in the past decade, mostly in finfish, and to a lesser degree in shrimp. The prebiotics are typically carbohydrates derived from plant or yeast origins, and comprised of three to ten carbohydrate units. Among those, inulin, fructooligosaccharide (FOS), mannanoligosaccharide (MOS), isomaltooligosaccharide (IMO) showed prebiotic characteristics in shrimp, as studies are summarized in the Table and more details explained in the text below.
The beneficial health effects of prebiotics are presumably due to the byproducts generated from their fermentation by gut commensal bacteria, which lead to promote the growth of health promoting bacteria and suppress the effects of harmful bacteria. Furthermore, some carbohydrates, such as short chain FOS (scFOS) and MOS could also be considered as immunosaccharides as they may directly activate certain innate immune responses, thus improving the host’s health. It should be noted that β-glucan molecules, well accepted non-specific immunostimulants, are not categorized as prebiotics in shrimp because crustaceans can digest glucan and use it as an energy source.
Shrimp solely depend on the innate defense system to defend themselves against the invasion of foreign micro-organisms as they don’t process the adaptive immune system. The innate immune system, also known as natural or non-specific defense system can be categorized in cellular and humoral reactions. Cellular components are directly performed by hemocytes, and include phagocytosis, encapsulation and nodulation. Humoral defense refers to processes related to activation comprised of the prophenoloxidase (proPO) system, the clotting cascade, a wide array of antimicrobial peptides, free radicals, and the synthesis and release of several immune proteins, such as antimicrobial peptides, proteinase inhibitors, cytokine-like factors, and others.
Inulin belongs to a class of dietary fibers known as fructans, composed of a polymer of β-D-fructose (F) attached by β -2-1 linkages. The first monomer of the chain is either a ß -D-glucopyranosyl or β -D-fructopyranosyl residue. D-fructose (F) link with D-glucose (G), with general structure of GFn. “n” refers to the degree of polymerization of inulin, and it’s usually 10 or so. Inulin is used by some plants as a means of storing energy and is typically found in roots or rhizomes. They constitute a group of oligosaccharides derived from sucrose that are isolated from natural vegetable sources.
Although inulin is not known as a natural fiber in shrimp diets, it has been suspected to contribute to balanced gut bacteria development, suppressing pathogens’ presence and effects. Bifidobacteria, lactic acid bacteria, and clostridia are known to be able to ferment inulin. A recent study showed that dietary supplementation of inulin decreased the prevalence of WSSV in Litopenaeus vannamei and increased the phenoloxidase activity, but didn’t affect hemocyte number, growth, survival, and lactic acid bacteria in shrimp. It seems unclear whether inulin alone can act as an immunostimulant or can work through its fermented by-products, such as short chain FOS, so that certain immune defense activities occur in shrimp.
Fructooligosaccharide is an inulin-like ingredient, having the same general formula of GFn, with n ranging from 1 to 5. Two studies evaluated dietary effects of scFOS in recirculating systems using different sizes of L. vannamei juveniles (0.17 g versus 7.5 g), and both found that gut microbiota was affected by dietary supplementation of scFOS. It was reported that scFOS improved specific growth rates and feed conversions in younger shrimp juveniles, and significantly affected the counts of Vibrio parahemolyticus, Aeromonas hydrophila, Lactobacillus sp. and Streptococcus faecalis in shrimp’s gut. Although another study using shrimp of bigger size observed no effect of dietary scFOS on weight gain, feed conversion or survival after a six week feeding trial, enhanced hemocyte respiratory bursts were observed in addition to increased colonization of a couple of gram positive aerobic microbes (Alkalibacillus spp. and Micrococcus spp.) and an unidentified seawater bacterium occurring in the digestive tract. However, whether these microbial shifts have any positive effects on the shrimp health, and through what mechanism, remains to be further investigated.
Mannan oligosaccharides, linear chains of mannose, are either derived from the cell wall of yeast (Saccharomyces cerevisiae) or plant mannans. The former consists of the chain of mannose residues linked together via α-1, 6-glycosidic bond, and the latter is β-1, 4-MOS produced by enzymatic hydrolysis via α-mannanases. In the past few years, several studies have been conducted to investigate MOS application in shrimp aquaculture and relatively consistent beneficial effects of dietary MOS were demonstrated, such as improving shrimp growth, feed efficiency, pathogen protection, intestinal microbiota modulation and functionality, etc.
MOS could affect the bacterial attachment in the intestinal tract, and a C-type lectin possessing mannose receptor was found in shrimp. MOS was shown to increase intestinal Lactobacilli and Bifidobacteria and reduce pathogenic Vibrio in the shrimp. The combined effects of MOS are likely due to the agglutination or binding of Vibrio cells to MOS, mediated by the presence of mannose receptors or possible interaction with other cagonical pattern recognition receptors to induce intracellular signaling pathways, followed by some innate immune responses being stimulated. More research is necessary to gain understanding of modes of action by MOS as prebiotics and immunostimulants.
Isomaltooligosaccharides, specifically, are mixed glucose oligomers with α-D-(1,6)-linkages. Dietary inclusion of 0.2% isomaltooligosaccharides alone showed no beneficial effect on shrimp performance, immune response and disease resistance. However, positive synergistic effects on shrimp immune responses and disease resistance was indicated when the combination of 0.2% IMO and 108 CFU/g Bacillus OJ was administered in L. vannamei through feed.
In summary, prebiotics showed some beneficial effects in shrimp aquaculture such as promoting growth, improving feed efficiencies, increasing disease resistance, etc. However, little is known in terms of mechanisms underlying some beneficial effects of prebiotics, synbiotics, and relationships with other immunostimulants.
Baseline information of shrimp digestive microbiota is critical in assessing the effectiveness of prebiotics supplementation, but such information has been lacking in general. Recently, Tucz and his co-authors isolated 64 bacterial strains with proteolytic activities from the digestive tract of L. vannamei, and identified the strains by combined molecular methods with phenotypic criteria as Pseudoalteromonas and Vibrio genera.
The adoption of advanced molecular tools and standardized methods in profiling gut microbiota in response to prebiotics, such as transcriptome and proteome profiling, would be helpful in deepening our understanding of various functions of prebiotics in microbial community shifts. RNA interference technology could also be a useful tool in investigating prebiotics- associated immune responses. Therefore, more detailed and in-depth research efforts in prebiotics are warranted to gain more understanding of their application to address the needs of promoting health and production in shrimp aquaculture.
Hui Gong, PhD, is an Associate Professor at the College of Natural and Applied Sciences at the University of Guam. Her expertise in shrimp aquaculture has built on 17 years of experience in applied research in both academic and industrial backgrounds.