Aquaculture industry produces the enormous amount of sea foods (fish, shrimp, planktons, etc.) with enriched quantity of proteins, essential amino acids, essential fatty acids, and micronutrients and also possesses the medicinal values. This production industry is very important to meet out the need of the global population. Recently, different culture practices for aquatic culturing organisms were developed in practices, where the risk of infection and diseases outbreak also increased which leads to the production loss to the aquatic sector. Several conventional methods are used to prevent the diseases probiotics, antibiotics, plants, immmunostimulants, proteins, immune proteins enhancement, nanoparticles, etc. At the same time, these treatment techniques also have merits and demerits to execute into the practical platform. For instance, chemical or antibiotics treatment into the culture system leads to the some adverse effects in culturing organisms, environment, and also consumer. In this chapter, various diseases caused by the bacterial strains and its control strategies in the shrimp farming industry to enhance the aquaculture are discussed.
- disease management
Shrimp farming plays the major role in aquaculture industry globally; due to its proteinaceous nature increased export viability and high profit yield the enhanced economy to the country. Penaeid (Rafinesque, 1815) shrimp aquaculture is one of the major industries which have rapidly grown during the past three decades in tropical and subtropical areas of the world (FAO 2019). Global production of shrimp increased from 1,564,563 metric tonnes in 2017 to 2,002,449 metric tonnes in 2019 (FAO 2019). The black tiger shrimp,
The shrimp immune system, like other invertebrates lacks an adaptive immune system and relies solely on its innate immunity against invading pathogens. Innate immunity is an ancient protective mechanism that appeared early in the evolution of metazoans and is divided into humoral and cellular responses , which work in jointly coordination for the detection/elimination of all foreign organisms potentially hazardous for the host . The cellular response mediated by haemocytes in hemolymph involves nodule formation, phagocytosis, encapsulation of pathogens and coagulation [4, 5]. The humoral components include the activation and release of molecules stored within haemocytes, such as anticoagulant proteins, agglutinins, phenoloxidase enzyme, antimicrobial peptides and protease inhibitors .
2. Commercially important shrimps in India
The Indian white shrimp,
In penaeid shrimp farming bacterial diseases are commonly associated with natural microbial flora of seawater, which possess enriched organic matter that supports the growth and multiplication of bacteria and other microorganisms. The most common shrimp pathogenic bacteria belong to the genus
3. Bacterial Septicaemia (
Acute hepatopancreatic necrosis disease (AHPND) is one of the severe systemic diseases caused by bacteria
3.1 Luminescent bacterial disease
This bacteria also causes the dangerous problems in aquaculture farms and heavy losses due to its infections it leads to the economy downfall and production rate loss too. These luminescent bacteria infected shrimps could be look like a fluorescent or luminescent producing nature in darkness.
3.2 Brown spot disease (Shell disease or rust disease)
Infected animals showed the brown and black erosions on the surface of the body and whole body appendages, this could be caused through
3.3 Necrosis of appendages
The tips of walking legs, swimmerets and uropods of affected shrimp undergo necrosis and become brownish and black. The setae, antennae and appendages may be broken and melanised. The epibiotic bacteria such as
Vibriosisin shrimp larvae
The affected larvae show necrosis of appendages, expanded chromatophores, empty gut, absence of fecal strands and poor feeding. Cumulative mortalities may be very high reaching up to 80% within few days.
3.4 Filamentous bacterial disease
The affected shrimp larvae show fouling of gills, setae, appendages and body surface. Molting of affected shrimps is impaired and may die due to hypoxia.
4. Control measure of shrimp disease
Most common pathogenic bacteria of penaeid shrimp include
Antibiotics are potential molecules for the initial treatments, though it has its own demerits such as continues usages of antibiotics in the environment like farms, aquatic systems might be causes the pollution and also leads to the development of multiple drugs resistant strains in the environment . For instance, adequate usage of chloramphenicol in shrimp farming sector in Myanmar, India, Pakistan, and Vietnam, paves the way to abuse of drugs resulting heavy loss in farming sector . 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 farmers . Use of microbes for beneficial purposes is increasingly recognized as a valuable input for sustainable aquaculture. Nowadays, several environmental-friendly prophylactic and preventive methods like probiotics, immunostimulants, antimicrobial peptides and quorum sensing interference are developed to control aquatic organism diseases. Therefore, novel antimicrobials with increased potency and least residual accumulation in shrimp tissue are required in lieu of conventional antibiotics for the management of bacterial epizootics. To keep the shrimp farming as a sustainable venture, new health management strategies must be used instead of the traditional methods like the abuse of antibiotics and chemotherapeutics.
5.1 Herbs as antibiotics
Herbs act as antibiotic for controlling or reduce the infection of pathogen in aquaculture sector and also increase the survival rate of organisms, during outbreak of disease managements. In
Vaccination is the practice of administering weakened or dead pathogenic bacteria, in order to confer long lasting protection through immunological memory . Adaptive secondary memory immune response of vertebrates depends on immunoglobulins (Igs), T-cell receptors (TCRS), major histocompatibility complex (MHC) and memory T cells. Memory cells and adaptive immunity differentiates the vertebrate and invertebrates immunity. Hence the several strategies are used to improve the adaptive immune system of invertebrates. Vaccination strategy must be designed with the key considerations of minimizing immunomodulatory stresses and stimulates the host defenses by triggering specific immune responses against infectious diseases.
Immunostimulants are chemical or natural source compounds that activate the immune system of aquatic animals and make them more resistant to infections by viruses, bacteria, fungi, and parasites. Stimulation of the non–specific immune system can improve the animal’s response to challenges from pathogenic bacteria. Immunostimulants used to control vibriosis in shrimp increased the survival rate [16, 17]. The potential of immunostimulants is to reduce the effects of bacterial diseases and to improve larval growth. Nowadays commercial immunostimulants are produced in the aquaculture sector to reduce the microbial diseases, through potential activity, immunostimulating performance are not in satisfied level. Immuno stimulation might be too drastic and harm or even kill the host. Because there is no memory component involved, the response is likely to be short in duration, and hence immunostimulants have to be administered repeatedly. In addition, long term administration of such agents seems to decrease the immune stimulatory effect and does not always promote disease resistance . Several bioactive compounds are isolated through various marine animals’ body components (Figure 2).
During the past two decades, the use of probiotics as an alternative to antibiotics has shown to be promising in aquaculture, particularly in fish and shellfish larviculture hatcheries . Probiotics could be used for the inhibitory studies because of its versatile nature such as inhibitory compounds production, competition for nutrients, competition for adhesion sites in the gastrointestinal tract, enhancement of the immune response, production of essential nutrients such as vitamins, fatty acids, and enzymatic contribution to digestion [20, 21]. Bacteria that are able to improve the water quality by removing toxic inorganic nitrogen or by mineralizing organic matter are also considered as probiotics. Bacterial strains dominantly present in culture water at high densities are also assumed to have the ability to compete efficiently for nutrients with possibly deleterious strains . The development of drug resistant bacteria and the reduced efficiency of antibiotic resistant for human and animal diseases, have led to suggestions of the use of nonpathogenic bacteria as probiotic agents to control diseases (Figure 3).
Recently, bacteriophages (phages) are proposed as candidate therapeutics for aquaculture . They could reduce pathogenic bacteria safely, effectively, and ecofriendly, as they are the natural enemies of bacteria. A major advantage of phage therapy is that non–target microbiota is not affected because the phages usually have a narrow host range . However, phages can transfer virulence factors rapidly and selective pressure on the
10. N-acyl homoserine lactonase
The first AHL-degrading enzyme was identified from Bacillus sp. expression of its gene in
11. The prophenoloxidase activating system (proPO system)
The proPO system is an efficient part of the innate immune response and consists of several proteins, which are involved in pattern recognition proteins, proteases, protease inhibitors, antioxidants proteins and melanisation as represented in Figure 4.
In addition, it also associated with the cytotoxic reactions, cell adhesion encapsulation, and phagocytosis, which is present in many invertebrate groups, such as ascidians, mollusks, echinoderms, millipedes, bivalves, brachiopods and insects [54, 55]. In invertebrates the humoral mediated immune system is triggered through several hemolymph proteins amongst the prophenoloxidase plays the vital role against the invading pathogens. At the same time, this immune pathway is stimulated through microbial pathogens such as bacteria, fungi and virus. The stimuli are derived from the outer membrane components of microbes, those molecules are termed as pathogen-associated molecular pattern (PAMP) which are lipopolysaccharide (LPS) and peptidoglycans (PG) from bacteria and β-glucans from fungi. This proPO cadcade consist of pattern-recognition proteins (PRPs) including LPS and β-1,3-glucan-binding protein (LGBP), β-1,3 glucan binding protein (βGBP), and peptidoglycan binding protein (PGBP), several serine protease and zymogens, proPO as well as proteinase inhibitors, which are important regulatory factors to avoid activation of the system where it is not appropriate .
12. Protein mediated nanoparticles
Alternative approaches to treat bacterial infections are urgently needed in aquaculture worldwide. Nanobiotechnology and nanotechnology products have a wide usage potential in aquaculture and seafood industries. For instance, production of more effective fish feed for aquaculture species by the application of nanotechnology is possible. New materials obtained by the nanosciences can be used in the different aspects of fisheries and aquaculture. Nanotechnology may have the potential to provide aquaculture that is safe from disease and pollution. Use of quorum quenching enzymes as antimicrobial agents is nature-inspired and has recently attracted much attention as an antibiotic-free approach to treat bacterial infections. The use of antimicrobial enzymes covalently attached to nanoparticles is of special interest because of enhanced stability of protein-nanoparticle conjugates and the possibility of targeted delivery.
13. Antimicrobial peptides
AMPs are effectors of the innate immune system and function as a first line of defense to fight against invading microorganisms  are represented in Figure 5. Therefore, AMPs are critical for shrimp to fight against the pathogenic invasion. AMPs are typically small in size, are naturally derived or synthetic and are active against a wide range of microorganisms, such as bacteria, virus, yeast, parasite and fungi, and they may also exhibit an anti-tumor activity [57, 58]. Generally, it has less than 150–200 amino acid residues, and it has an amphipathic structure with cationic or anionic properties. Several families of shrimp AMPs, such as penaeidins, lysozymes, crustins, ALFs and stylicins, have been identified and characterized [59, 60]. They are produced by and stored in the hemocytes; these are key cells in the crustacean immune system . Various methods are discussed in the introduction section to eradicate the bacterial inhibition and bacteria causing disease management. However, in this chapter, we used the antimicrobial peptides to inhibit the bacterial causing biofilms and using probiotic bacteria, we attempted to reduce the bacterial disease.
Crustins are generally defined as multi-domain cationic antibacterial polypeptides (7–14 kDa) containing a whey acidic protein (WAP) domain at the C-terminus (Figure 5) . The first identified crustin member is an 11.5 kDa protein purified from the granular haemocytes of the shore crab,
14. Materials and methods
14.1 Collection and maintenance of bacterial strains
14.2 Replica plating method
The shrimp intestinal tract, hepatopancreas content was aseptically removed from a live healthy prawn were homogenized and serially diluted with sterilized normal saline solution. Suspensions (0.1 ml) were spread on different media like nutrient agar (with 1% w/v NaCl) and thiosulfate/citrate/bile/salt (TCBS), Zobell Marine agar (ZMA), bacillus medium (HiMedia, India), and incubated under an aerobic atmosphere overnight at 37°C for 24 h. After incubation, predominant bacterial colonies were selected based on their morphological characters including color, shape, size colonies from all media were replica plated on the Muller Hinton agar medium (Hi Media, India) with target bacterial strains and incubated at room temperature for 24 h. After incubation viable colonies showing the zone of clearance against the target
14.3 Antagonism assay
To evaluate the potential antagonistic activity of the isolated probionts by well diffusion assay on solid medium and eight
14.4 Characterization of strains
Isolated strains were subjected to standard morphological, biochemical assay followed by bacterial genomic DNA was extracted using the method . Bacterial strains were cultivated in 10 mL of Luria-Bertani broth (LB) at 29°C in agitation for 18 h. culture was centrifuged for 5000 rpm for 5 min suspended pellet with Sucrose TE buffer 10 mmol−1 lysozyme was added and incubated for 30 min. After incubation, add 100 μl of 0.5 M EDTA (pH 8) and 60 μl of 10% SDS added with 250 μl of equilibrated Phenol and 250 μl of Chloroform and mixed gently mixture was centrifuged. An equal volume of chloroform and isoamyl alcohol mixture (24:1) was added with shaking the mixture. Collect the aqueous phase in a sterile tube and precipitate it with 2 volumes of 100% ethanol and 3 M Sodium Acetate. Store at −20°C for 30 min. Followed by this addition, the sample was centrifuged with at 12000 rpm and the isolated DNA was precipitated with 70% ethanol. DNA was suspended with 30 μl of TE Buffer pH (8.0) and DNA was extracted for 16S rRNA sequence determination & RAPD analysis.
14.5 rRNA gene amplification
The region of the 16S ribosomal gene (rRNA) of the DNA extracted from each bacterial strain was amplified by the polymerase chain reaction (PCR). The reagent mixture was prepared with the universal 16S rRNA Fp 5′-AGA GTT TGA TCC TGG CTC AG-3′ and 16S rRNA Rp 5′-ACG GCT ACC TTG TTA CGA CTT-3′ , samples were amplified by PCR in Std buffer 2.5 μl, dNTPs 0.5 μl, forward and reverse primers each 1.0 μl and Taq 0.2 μl and template DNA 1.0 μl condition consist of 40 cycles of 95°C (5 min), 55°C (1 min), and 72°C for (2 min) and with final 72°C for 10 min for elongation process were performed with four bacteria strains, yielding positive amplification for all DNA tested, as determined by visualization on agarose gel electrophoresis. The amplification products were purified by using Real genomics kit, by following the specifications of the manufacturer.
14.6 Co-culture method
The co-culture method was performed to observe the antagonistic potential and reproductive effect of the isolated bacteria, when grown with the
14.7 Artemia hatchability test
Probiotic activity of chosen strains were further tested with Artemia hatchability test, 160 mg of dried cysts were hatched in 80 ml of sterile sea water under the conditions of strong aeration and constant illumination, at 28°C
where N = Nauplii, C = Unhatched cysts, and U = Umbrella stage.
14.8 Challenge studies in
Five experimental groups namely
The active nauplii were considered as live and counted under microscope.
Various approaches are applied to eradicate the bacterial and other microbial diseases in aquaculture, however this chapter deals with the biofilm inhibitory mechanism through two different ways probiotic isolation from aquaculture farms or the specific animals such as shrimp or crab form the body part. For this many microbiology techniques are used to isolate the bacteria and identify, then inhibit the
16. The potential use of antimicrobial peptides for disease control in aquaculture
Antimicrobial peptides provide a good therapeutic alternative for the treatment of diseases in aquaculture. Several antimicrobial peptides from various sources are already in clinical and commercial use . However, it is quite promising that the shrimp AMPs could be potential candidates as an alternative to antibiotics in shrimp farming. Besides their antimicrobial function, AMPs are also known to act as mediators of inflammation influencing diverse processes such as cell proliferation, wound healing, cytokine release and immune induction .
The significance of aquaculture in the context of global food production sector, the management of aquatic resources and the socio-economic development of coastal rural areas is now fully appreciated world-wide. In the last decade, a series of papers describing shrimp immunity were published and a batch of related data accumulated, which are very useful for understanding the interaction between shrimp and pathogens to enrich the immune theory of invertebrates. Recently, several review papers summarize the achievements in shrimp immunity including expressed sequenced tag and database construction , microarray analysis of shrimp immune response , shrimp molecular responses to viral pathogen  and the cationic antimicrobial peptides in penaeid shrimp . Obviously understanding the shrimp immunology is necessary to develop an effective strategy for disease control. Indian white shrimp
Recent advances gives the notions to incorporate the different techniques and utilized in the aquaculture practical system as a combined mode to enhance the potential activity of the strategies towards the microbial pathogens, this would be efficient method when compare to conventional techniques alone. In addition this way of microbial eradication will helps to improve the aquaculture production as well as cost effective.
The study was supported by the National Research Foundation of Korea, which is funded by the Korean Government [NRF-2018-R1A6A1A-03024314].
Conflict of interest
The authors declare no conflict of interest.