Shrimp aquaculture is constantly threatened by recurrent outbreaks of diseases caused by pathogenic bacteria of the genus Vibrio. Acute hepatopancreatic necrosis disease (AHPND) is one of the most aggressive vibriosis reported to date in the shrimp industry. AHPND provokes massive mortalities, causing economic losses with strong social impacts. Control of vibriosis requires the application of multifactorial strategies. This includes vibrio exclusion, shrimp microbiota, particularly in the digestive tract, and shrimp health management through immune stimulation. This paper reviews these two strategies for the prophylactic control of vibriosis. First, we describe the devastating effects of AHPND and the cellular and humoral effectors of the shrimp immune system to cope with this pathology. Secondly, the mechanisms of action of probiotics and their positive impacts are highlighted, including their immunostimulant effects and their role in the balance of the shrimp microbiota. Finally, we reviewed immunostimulants and prebiotics polysaccharides that together with probiotics act benefiting growth, feed efficiency and the microbiota of the digestive tract of farmed shrimp.

Keywords: Penaeus vannamei, Vibrios, probiotics, immunostimulant, prebiotics.

Penaeus vannamei shrimp aquaculture is one of the fastest-growing aquaculture sectors worldwide¹. In Latin America, the productions of Ecuador, Mexico, Honduras and Brazil stand out². However, outbreaks of infectious diseases caused by pathogenic agents such as viruses and bacteria^3–5 limit the industrial development of shrimp farming. Among the most aggressive viral diseases that have attacked farmed shrimp are white spot syndrome (WSSV)^6–8 and Taura syndrome (TSV)^9,10. Unlike viruses, bacteria, can remain indefinitely in culture systems because of their ability to produce biofilms⁵. Vibrios spp are the most aggressive bacteria for farmed shrimp, causing recurrent and emerging diseases^11–13.

Strains of Vibrio parahaemolyticus and other vibrio species carrying the toxin, PirA and PirB are highly virulent for penaeid shrimp^14–16 because they can cause acute hepatopancreatic necrosis disease (AHPND)^17–19. AHPND causes significant losses in the shrimp industry globally and socioeconomic impacts20 that are estimated at more than $1 billion per year²¹. V. parahaemolyticus is a marine microorganism, which can grow in estuarine waters with tolerance to high temperature, high pH and high salinities with an affinity for marine plankton^22,23. The success of vibrios is based on their ability to grow in shrimp culture systems characterized by high temperatures, high salinity ²⁴ abundant presence of exuviae and high organic matter load. Under these conditions, vibrios thrive and modulate the microbiome at their advantage²⁵.

Among the aspects that should be considered in closed systems designed to prevent the entry of vibrios and other potential pathogens are adequate nutrition with functional additives to improve the immune response^26,27 and biosecurity controls²⁸. In semi-extensive cultures, which involve earthen ponds in estuarine environments biosecurity is not sufficient and other alternatives must be considered to protect shrimp against vibriosis²⁸. Among these alternatives, there are anti-virulence therapy ^29,30, probiotics^31,32 and immunomodulation^33,34.

The main objective of probiotics is to exclude pathogenic bacteria from culture systems. In addition, probiotics enhance host growth and health by improving stress resistance ^35,36 and promoting better nutrient utilization in shrimp by improving feed digestibility ^37,38. Despite their effectiveness, the application of living organisms faces several challenges due to the preparation, bioactivity, stability, and success in different aquaculture environments. Another promising strategy is the enhancing of immune defenses. Immuno-stimulation seeks to activate the shrimp immune system based on the use of pathogen-associated molecular patterns (PAMPs) or molecules that resemble them and the recognition by their receptors in the host^39,40, thus increasing immune resistance. Different polysaccharides fall in this category, among these, β-glucans and peptidoglycans⁴¹. The synergistic use of probiotics and immunostimulants ^26,42 or the use of polysaccharides as prebiotics that help the establishment and aptitude of probiotics^27,43 are also explored to face vibriosis. In this review, we present the devastating effects of AHPND and the cellular and humoral effectors of the shrimp immune system to cope with this pathology. We also report the strategies to control vibriosis in farmed shrimp, based on probiotics and immunostimulants.

Diseases produced by species of the genus Vibrio have caused significant economic losses in the shrimp industry worldwide^20,44. Belonging to the phylum Proteobacteria, class Gammaproteobacteria, the genus Vibrio of the family Vibrionaceae includes 65 well-documented species of heterotrophic bacteria⁴⁵. Vibrio harveyi and V. parahaemolyticus belonging to the clade Vibrio harveyi are among the most critical pathogens for shrimp46. Vibriosis cause massive mortality rates and can affect 100% of production^8,12,47. Vibrios cause tail necrosis, loose shell syndrome, white gut, septic hepatopancreatis, systemic and wound vibriosis, causing hypoxia and death of shrimp from hatchery tanks to growing ponds¹².

AHPND, the most aggressive vibriosis recorded to date, was detected in China in 2009 and other Southeast Asian countries⁴⁸. In 2013, this disease was reported in Mexico 18 and spread to South America ^49,50. PirA and PirB toxin genes were detected in other vibrio species of the V. harveyi clade51, Vibrio owensii⁵², Vibrio Campbelli ⁵³ and recently in a Vibrio not belonging to this clade, Vibrio punensis⁵⁰. The observed damage to shrimp health by AHPND has been devastating. Vibrios initiate their attack by colonizing the stomach14 of infected shrimp, originating dysbiosis ^25,54 and generating toxins that subsequently cause hepatopancreatic cell damage¹³ and eventually hepatopancreatic septicemia ^5,17. Shrimp affected by AHPND show severe necrosis in the hepatopancreas with massive detachment of epithelial cells and total loss of tissue structure. Pale atrophied hepatopancreas, intestinal epithelial necrosis and massive infiltration of hemocytes are observed⁵⁵. It has also been reported that V. parahaemolyticus strains carrying PirA and PirB toxin genes alter the microbiome of infected shrimp, decreasing bacterial diversity and increasing the fitness of vibrios to proliferate in culture environments^25,56.

Shrimp, like all crustaceans, possess an immune system that effectively protects them from harmful microorganisms⁵⁷. In penaeid shrimp, two lines of immune defense can be distinguished. The first line is a physical barrier formed by the cuticle which is mainly composed of polysaccharides, chitin, and mineral deposits⁵⁸. Internally, contact with the medium is very limited due to the presence of cuticle in the stomach⁵⁹, the constantly growing peritrophic membrane lining the epithelium of the midgut and hindgut ⁶⁰ and the gastric sieve that limits passage into the hepatopancreas to particles smaller than 1 μm. The second line of defense is its innate immune system. It is based on humoral and cellular effectors, being hemocytes the main effectors of immunity. These effectors act together to eliminate infectious microorganisms⁶¹.

Innate immunity with specificity or immune priming has also been described in shrimp and other arthropods. This is based on the Down syndrome cell adhesion molecule (Dscam), a hypervariable recognition protein produced by alternative splicing. Dscam may be involved in both innate immunity and pathogen-specific immune response⁶². Shrimp cells innately recognize invading microbes through pattern-recognition proteins (PRRs) that act as pathogen-associated molecular pattern receptors (PAMPs)⁶³. Innate immune cells respond rapidly to this activation through intracellular signaling cascades leading to humoral and cellular immunity activation ⁶⁴.

Humoral effectors involve the synthesis and secretion of immune proteins from hemocytes into the circulatory system or hemocele⁶⁵ and the release of enzymes of the prophenoloxidase (proPO) cascade leading to the generation of oxidative metabolites and deposition of toxic melanin⁶⁶. Pathogens are recognized by pattern recognition proteins that recognize lipopolysaccharide (LPS) and β-1,3-glucan (BGBP)^67–69, initiating the chain of proPO activation, culminating in melanization of the wound or intruding organisms^70. Another mechanism of humoral response is the clotting system. The clotting cascade involves both hemocytes, which release transglutaminase⁷¹, and the coagulation factor present in the plasma72. Released transglutaminase polymerize the plasmatic clotting factor⁷².

Upon microbial challenge, hemocytes release antimicrobial peptides (AMPs)⁷³. AMPs are cationic or anionic proteins that attack invading microorganisms ^65,74. AMPs are small, from 15 to 100 amino acids and amphipathic in structure⁷⁵. They can rapidly diffuse to the point of infection and destabilize the membrane of microorganisms, acting as antibiotics against gram-negative and gram-positive bacteria76 and against yeasts and filamentous fungi⁷⁷. It has also been reported that AMPs modulate immunity^74,78. In Crustacean the main AMPs are represented by the three families of cationic peptides: penaeidins, crustins and antilipopolysaccharide factor (ALF)^79,80. Shrimp AMPs can be classified as cationic and anionic, the former being more abundant. Only stylicin, a peptide derived from hemocyanin and some ALFs are anionic AMPs65. Each family of shrimp AMPs shows sequence diversity and constitute multiple classes, isoforms, or subgroups⁷⁵.

Penaeidins have affinity for shrimp chitin, being detected in the cuticle after microbial challenge^73,81. Penaeidin mRNAs are present in all larval stages from nauplius onwards but vary in expression levels82.  In penaeid shrimp, crustin homologs have been primarily identified by expressed sequence tag (EST) analysis of cDNA libraries from hemocytes of P. vannamei, P. setiferus83, and P. monodon^84,85. Crustin is the largest AMP family reported in crustaceans86. In shrimp, most crustins are constitutively expressed in hemocytes but not in the hepatopancreas^80,87,88. Some P. monodon crustin isoforms are expressed primarily in tissues other than hemocytes. ALFs are 10 to 14 kDa molecules. In shrimp, they were first identified in hemocytes of P. setiferus87 and P. monodon_84,89. ALFs exhibit a potent minimum inhibitory concentration (MIC) (<6.25 μM) and spectrum of antimicrobial activity, covering a large number of gram-positive and gram-negative bacteria, filamentous fungi and even enveloped viruses^90,91.

A lower-than-normal number of circulating hemocytes in crustaceans correlates with reduced resistance to pathogens⁹⁷, while an increase in the number of hemocytes enhances the immune response during periods of stress, increasing resistance to diseases ^98,99. In addition, wound repair processes in shrimp are influenced by the concentration of hemocytes and penaeidins released by them in the wound area to eliminate invasive pathogens and improve hemocyte adhesion¹⁰⁰. The open circulatory system of shrimp¹⁰¹ allows hemocytes to infiltrate and adhere to many tissues through the interstitial spaces of different organs and tissues without being limited to the vascular system¹⁰².

Hemocytic nodules, detected in connective tissue (Figure 1B), gills and hepatopancreas, are formed by numerous hemocytes that act synergistically to trap microorganisms or large antigens that cannot be eliminated by phagocytosis. These nodules undergo subsequent activation of the proPO system, with consequent melanization and destruction of microbes¹⁰³ , such as the hemocytic nodules observed in tubules of the hepatopancreas of shrimp infected by vibrios^18,104. If the lesions are very large, they will form hemocytic capsules like those observed around hepatopancreatic tubules injured by vibrios¹⁰⁵. In shrimp, typical inflammatory response reactions with hemocyte infiltration and melanin deposition in nodules and capsules are observed during bacterial infections¹⁰⁶ (Figure 2A). The respiratory burst, the mechanism that leads to the generation of reactive oxygen intermediates, is another microbicidal process used by shrimp hemocytes in phagocytosis and encapsulation107

In penaeids shrimp, some of the defense functions are performed in the lymphoid organ (LO), which actively participates in capturing, isolating, and eliminating pathogens by phagocytosis^108,109. The LO is a bilobed structure located in the anteroventral region of the hepatopancreas and posterior to the antennal gland, being an integral part of the distal portion of the subgastric artery^110,111. The LO is considered an integral part of the circulatory system of penaeid shrimp and is believed to work as a hemolymph filter^110,112. In histological sections it is seen as a vascularized tissue surrounded by hemal sinuses. The LO tubules have an inner layer of endothelial cells, surrounded by stromal tissue, bound by connective tissue^109,110,113. In the presence of natural or experimental infections (mainly of viral etiology), nodular hyperplasia known as lymphoid organ spheroids (LOS) form (Figure 2B). These structures have also been detected in the connective tissues of other organs of shrimp and are called ectopic spheroids^114,115. It has been reported that the LO filters viral particles of TSV¹¹⁴ and WSSV116. In late stages of infection, viral particles are detected in the spheroids of the lymphoid organ, whose cells enter apoptosis^114,116. In addition to LOS, apoptosis has been reported in viral infections as a control mechanism of viral replication^114,116. Other tissues involved in hemolymph clearance mechanisms are the heart reserve phagocytes, podocytes of the gills, and antennal gland¹¹⁷. The antennal gland is an important excretory organ in shrimp that is functionally similar to the kidney in vertebrates^118,119, and therefore exposed to external medium.

Vibriosis is the leading bacterial disease-causing mortality of cultured shrimp^120,31. Antibiotics and chemotherapeutic agents were the first tools to control vibriosis. However, antibiotics have led to the development of resistant microorganisms¹²¹ with risk to human health due to the horizontal transfer of resistance genes from shrimp vibrios to human pathogenic bacteria^122,123. The use of antibiotics also carries risks to the environment, due to the accumulation of environmental residues¹²⁴. In addition, excessive levels of antibiotics can deteriorate the general condition of shrimp¹²⁵ and cause rejection of the final product in international markets. Therefore, implementing effective and environmentally friendly multifactorial strategies for the control of vibrios in shrimp production systems is to be considered a priority¹²⁶. These include the anti-virulence strategy^127,128, which is based on blocking bacterial communication known as quorum sensing (QS), to inhibit the expression of virulence genes. The QS mechanism uses small chemical signaling molecules called autoinducers (AIs)^129,130. This strategy has been applied in aquaculture and proposed by several authors as a promising tool for the control of pathogenic bacteria ¹²⁸. Other alternatives to control vibriosis are the management of the microbiota, through probiotics  ^121,131,132 and immunostimulation^31,133(Figure 3).

The term probiotic was defined by Fuller (1989)¹³⁴ as «a live microbial food supplement that improves bacterial balance, benefiting the host’s health”¹³⁵. Probiotics emerge as a promising alternative to decrease the aquaculture industry’s dependence on antibiotics ¹³⁶. Probiotics have been applied in fish and invertebrate cultures¹³⁷, especially in South America, Ecuador, and Asia, particularly China and India. Probiotics for aquaculture belong mainly to bacteria of the genera Bacillus, Lactobacillus. Pseudomonas, and vibrio, in addition to yeasts^31,138,139.

In the field of aquaculture, the concept of probiotic is extended to “a live microbial supplement that has a beneficial effect on the host by improving the environmental microbial community, ensuring better use of the feed or its nutritional value, and improving the response to disease or the quality of culture environment”¹²¹. In addition probiotics can be applied directly to the culture water as bio remediators ^140,141, the modes of action of probiotics are diverse¹⁴² (Table 1). Applied in feed, probiotics improve digestion, enhance immune response, and inhibit the growth of pathogenic bacteria^143–145. Probiotics competitively exclude vibrios^26,146,147 and enhance shrimp immune indicators, phagocytosis, nodule formation, activation of the proPO system and generation of AMPs¹⁴⁸. Probiotics release several antibacterial agents with bactericidal or bacteriostatic properties that inhibit the colonization of pathogenic organisms in the shrimp gut1^49–151. Probiotics can modulate the expression of immune genes and gut microbiota during stress induced by high stocking density¹⁵².

Bacteria of the Bacillus genus are the most widely used in shrimp aquaculture as probiotics because they form spores, which facilitates conservation and commercial distribution. In addition, Bacillus bacteria are well perceived by the consumer because they are gram-positive bacteria. Among other advantages of Bacillus spp are their ability to grow using different carbon and nitrogen sources¹⁵³. Bacillus spp can grow in the marine environment and produce a variety of bioactive compounds that act on growth, health, disease resistance ^154,155 and culture water quality^156,157. It has been further reported that Bacillus spp. produce a wide range of antimicrobial peptides^158,159. Bacillus spp. supplementation decreases mortality in shrimp exposed to pathogenic Vibrios^160,161. The most used species are Bacillus subtilis and Bacillus lechiniformis. Other probiotics successfully used in aquaculture belong to other bacterial genera, including Vibrios¹⁶² or marine symbionts such as Pseudovibrio denitrificans¹⁶³. Vibrio diabolicus is a very effective probiotic controlling vibriosis affecting P. Vannamei in larval stages¹⁶⁴ and juveniles⁵⁶. Because it is a vibrio, this bacterium has the advantage of thriving in the culture conditions of shrimp. V. diabolicus can compete effectively with pathogenic vibrios⁵⁶, thus having positive effects on the immune system of shrimp^133,164.

Several aspects must be considered for selecting and using probiotics in shrimp. Among these are the ability to grow in marine environments, exclude vibrios, and compete with them for colonization of shrimp and surfaces of culture systems to which vibrios are well adapted. Bacterial antagonism is a common phenomenon in nature; therefore, microbial interactions play an important role in balancing beneficial competitors and potentially pathogenic microorganisms ³¹. In this sense the ability to compete and colonize is one of the most desirable properties of probiotics. There are several aspects behind the ability to compete and colonize one of them is competitive exclusion, which occurs when an established microflora prevents or reduces the colonization of a bacterial competitor for the same location¹⁴⁰.  For pathogenic bacteria the ability to adhere to host substrates is related to virulence and is considered as the first step for an infection¹⁷⁸.

Probiotics must have the ability to compete for mucosal attachment sites and competition for nutrients¹⁴⁰. Therefore, the attachment ability of probiotics is another important criterion for their selection and application in aquaculture^179,180. Colonization ability further depends on migration in a coordinated manner across organism surfaces¹⁸¹. For these processes, bacteria use cell appendages such as fimbriae and flagella, structures important for colonization and the creation of biofilms^182–184. By producing inhibitory substances, probiotics prevent pathogen replication and reduce pathogen colonization^132,140. Among the extracellular products that inhibit or kill other potentially pathogenic bacteria have been found: antimicrobial substances^185,186, organic acids¹⁸⁷, bacteriocins¹⁸⁸, or hydrogen peroxide. Another important aspect is the morphophysiology of shrimp. Indeed, the shrimp has an external and internal cuticle covering the stomach, which is lost during molting, and the peritrophic membrane in continuous growth, covering the midgut and hindgut⁶⁰. Therefore, probiotics with affinity for the cuticle and chitin, should be applied continuously.

The immune system can recognize and react to PAMPs through different PRRs¹⁸⁹.     Different PAMPs, such as LPS, peptidoglycans and β-glucans can activate the shrimp immune system, acting as immunostimulants. Immunostimulants are an effective alternative to prevent diseases in shrimp. There are several sources of these molecules in nature, including gram-negative bacteria (lipopolysaccharides), gram-positive bacteria (peptidoglycans), fungi, yeasts, and algae (β-glucans). The most used immunostimulants are β-glucans from different marine and terrestrial sources¹⁹⁰. The β-glucans are polysaccharides other than starch; they are present in the cell wall of bacteria, fungi, and plants. They are used to increase the resistance of shrimp against bacterial and viral infections ^26,191. The β-glucans extracted from different sources exhibit various immunomodulatory functions, acting as PAMPs, they interact with PRRs to activate the innate immune response¹⁹². In shrimp β-glucans extracted from yeast Candida parapsilosis has an immunomodulatory effect against WSSV, obtaining high survivals¹⁹³. In addition to their effects on the immune system, β-glucans from bacteria improve the nutritional and antioxidative status of shrimp¹⁹⁴. β-glucans from marine yeasts improve respiratory burst and phenoloxidase activity¹⁹⁵. The beneficial effect of β-glucans can be enhanced by degradation to obtain oligo β-glucans with improvements in their immunostimulatory and growth, promoting effect in shrimp¹⁹¹. The benefits of using β-glucans have been evidenced by successful results in survival and performance in shrimp ponds²⁶. Immunomodulatory effects on penaeid shrimp have been also reported for other polysaccharides that exhibit repeating molecular patterns, such as arabinose and galactose heteropolysaccharides from Angelica sinensis, and cocoa pectin¹⁹⁶.

As previously described, innate immunity with specificity or immune priming has been reported in shrimp and other arthropods¹⁹⁷. From the perspective of a possible pathogen-specific immune response, vaccination trials have been performed in shrimp. Thus in P. monodon the VP28 protein (WSSV) has been used as antigen, observing immunomodulatory effects on different immune effectors:caspase, penaeidin, crustin, astakine, syntenin, PmRACK, Rab7, STAT and C-type lectin¹⁹⁸.

Prebiotics are indigestible fibers for the host, but are digested by beneficial bacteria, which improve the health of the intestine^199,200 and can be referred to as functional saccharides²⁰¹. Moreover, prebiotics are associated with improvements in growth, feed efficiency, digestive enzyme activities, proper gut morphology, improved immune status and disease resistance^136,202,203. Prebiotics such as fructooligosaccharides (FOS), galactooligosaccharide, mannan oligosaccharides, and inuline are applied in shrimp culture²⁰⁴. The application of short-chain fructooligosaccharides (ScFOs) in P. vannamei diet increased the weight and specific growth rate of shrimp205. ScFOS supplemented at concentrations of 0.025 to 0.800 wt% increase the respiratory burst hemocytes206. More details about prebiotics in farmed shrimp are shown in table 2.

The survival rate, yield, size, color, and taste of shrimps are critical for its market. With the increased loss of worldwide production due to vibriosis and the risks inherent in the use of antibiotics, new products to control vibriosis must consider the need for “organic” shrimps without antibiotics. Among the most promising alternatives are probiotics. The modes of action of probiotics are diverse. They improve digestion, enhance immune response, and inhibit the growth of pathogenic Vibrio spp. Probiotics release several antibacterial agents with bactericidal or bacteriostatic properties that inhibit the colonization of pathogenic Vibrio spp in the shrimp. During the last years, new approaches have emerged to identify and isolate novel probiotics. One of them is the use of marine symbionts, but even if first results are highly encouraging, a deeper understanding of the biological mechanisms is still needed. Another aspect that is gaining relevance is the ability of probiotics to recover or maintain healthy microbiota, curbing dysbiosis, and restoring microbial balance. The application of living organisms faces several challenges, due to their differences in bio activity, and effectiveness in marine aquaculture environments. Another promising option is the use of polysaccharides that increase shrimp resistance to pathogenic Vibrio spp. by immune stimulation. Polysaccharides can also act as prebiotics, being used as energy sources by beneficial bacteria, which improve the health of the host intestine. The significant advances in combating the AHPND in the shrimp industry have evidenced several strategies to solve this critical challenge, but it is not yet clear which is the best of the previously mentioned in this review. It is likely that while each has its advantages and disadvantages, a possible solution could be a combination of them.

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Received: 23 October 2021 / Accepted: 15 February 2022 / Published:15 May 2022

Citation: Ramírez M, Debut A,  Alexis F, Rodríguez J. Control of vibriosis in shrimp through the management of the microbiota and the immune system. Revis Bionatura 2022;7(2) 1. http://dx.doi.org/10.21931/RB/2022.07.02.1