Shrimp disease management for sustainable aquaculture: innovations from nanotechnology and biotechnology

Abstract

Infectious diseases in shrimp are one of the significant constraints in shrimp aquaculture across the globe. The alarming increase of antibiotic resistance in bacteria has rendered antibiotic therapy a controversial subject today. Therefore, the practice of antibiotic therapy in aquaculture needs to be dissuaded and appropriate alternatives have to be found out. For the past few decades, the field of nanotechnology and biotechnology has proposed novel and effective solutions involving the use of nanoparticles, biofilm-based vaccines, algal extracts, phytobiotics, probiotics, prebiotics, and synbiotics to combat infectious diseases. Algal extracts, phytobiotics, probiotics, prebiotics, and synbiotics are compounds of biological origin due to which they are safe to use in aquaculture settings. With the advent of green and bio-nanotechnology, nanomaterials are also becoming a safer alternative to antibiotic therapy. Vaccines developed from antigenic components of bacterial biofilms are more promising than regular vaccines synthesized from antigens of planktonic forms. Some of these methods have extended applications in shrimp aquaculture in the form of immunomodulants, diagnostic tools, drug and vaccine carriers. The hazards of chemotherapy in shrimp aquaculture can be overcome by replacing antibiotics and other chemical agents with these new approaches. Adopting these strategies makes aquaculture-based food more organic, consumer-friendly and helps in establishing sustainable aquaculture. This review reveals the ill effects of antibiotic therapy in shrimp aquaculture and casts light on the advantages and the research gaps in these strategies that need to be addressed.

Graphical Abstract

Full text

Shrimp disease management for sustainable
aquaculture: innovations
from nanotechnology and biotechnology
P.S Seethalakshmi
1
&Riya Rajeev
1
&George Seghal Kiran
2
&Joseph Selvin
1
Received: 7 December 2020 /Accepted: 19 March 2021 /
#The Author(s), under exclusive licence to Springer Nature Switzerland AG 2021
Abstract Infectious diseases in shrimp are one of the significant constraints in shrimp
aquaculture across the globe. The alarming increase of antibiotic resistance in bacteria has
rendered antibiotic therapy a controversial subject today. Therefore, the practice of
antibiotic therapy in aquaculture needs to be dissuaded and appropriate alternatives have
to be found out. For the past few decades, the field of nanotechnology and biotechnology
has proposed novel and effective solutions involving the use of nanoparticles, biofilm-
based vaccines, algal extracts, phytobiotics, probiotics, prebiotics, and synbiotics to
combat infectious diseases. Algal extracts, phytobiotics, probiotics, prebiotics, and
synbiotics are compounds of biological origin due to which they are safe to use in
aquaculture settings. With the advent of green and bio-nanotechnology, nanomaterials
are also becoming a safer alternative to antibiotic therapy. Vaccines developed from
antigenic components of bacterial biofilms are more promising than regular vaccines
synthesized from antigens of planktonic forms. Some of these methods have extended
applications in shrimp aquaculture in the form of immunomodulants, diagnostic tools,
drug and vaccine carriers. The hazards of chemotherapy in shrimp aquaculture can be
overcome by replacing antibiotics and other chemical agents with these new approaches.
Adopting these strategies makes aquaculture-based food more organic, consumer-friendly
and helps in establishing sustainable aquaculture. This review reveals the ill effects of
antibiotic therapy in shrimp aquaculture and casts light on the advantages and the research
gaps in these strategies that need to be addressed.
Keywords Antibiotic resistance .Nanomaterials .Phage therapy .Phytobiotics .Probiotics .
Synbiotics
https://doi.org/10.1007/s10499-021-00698-2
Handling Editor: Brian Austin
*Joseph Selvin
josephselvinss@gmail.com; jselvin.mib@pondiuni.edu.in
1
Department of Microbiology, Pondicherry University, Puducherry 605014, India
2
Department of Food Science and Technology, Pondicherry University, Puducherry 605014, India
Published online: 14 April 2021
Aquaculture International (2021) 29:1591–1620

Introduction
Shrimp aquaculture is the practice of breeding, rearing, and harvesting of shrimp species in
man-made environments for human welfare. Shrimp is one of the most widely consumed
seafood around the world (FAO 2019). Considering the population increase, there is a massive
demand for shrimp-based food products and aquaculture plays a crucial role to meet them
(Kobayashi et al. 2015). The development of shrimp aquaculture has also decreased the
problems associated with overfishing in marine ecosystems (Pradhan and Flaherty 2007).
Greater acceptance of shrimp aquaculture at the global level animal-based food supply
indicates the necessity of sustainable development and extensive production of several shrimp
varieties (Rajeev et al. 2021).
Aquaculture practices have brought shrimp to artificial environments where they can
encounter various pathogens generally absent in their natural habitat (Nga et al. 2005).
Variations in pH of rearing water, temperature, dissolved oxygen content, and presence of
toxic chemicals are some of the commonly encountered physical and chemical factors that
contribute to poor growth and mortalities in shrimp (Nga et al. 2005). The chances of
infections emerge due to poor water quality and feed, which weakens the innate immunity
of shrimp. Mass mortalities in shrimp due to infections occur due to the transmission of
pathogens when densely stocked (Wiyoto et al. 2017). The exportation of farmed shrimp
commodities to many countries has also resulted in pandemics in aquaculture farms (Walker
and Mohan 2009). Diseases in aquaculture industries can be a threat for humans and other wild
aquatic species too, as pathogens can cross species barriers (Walker and Mohan 2009;Zhang
et al. 2018). Antibiotic therapy for disease prevention in shrimp aquaculture was initially a
huge success, until microorganisms started acquiring resistance against antibiotics. Antibiotic
resistance renders bacterial infections in shrimp recalcitrant to antibiotic therapy, and
antibiotic-resistant genes can get transmitted to nearby ecosystems, from shrimp farms. Human
pathogens receiving such antibiotic-resistant genes from aquaculture settings can challenge our
concurrent health system (Holmström et al. 2003). Hence, strategies safe for aquatic life,
humans, and the environment need to be implemented. Research in nanotechnology and
biotechnology have experimented and analyzed holistic approaches that serve as a potential
alternative to antibiotics in controlling infectious diseases in shrimp aquaculture. This review
deliberates the issues of antibiotic usage in the aquaculture sector and the benefits of
environment-friendly approaches such as algal extracts, phytobiotics, phages, biofilm-based
vaccines, probiotics, and synbiotics.
Infectious diseases and the advent of antimicrobial resistance in shrimp
aquaculture
Infectious diseases have affected shrimp aquaculture industries drastically, and the majority of
infections in cultured shrimp are of bacterial and viral origin with few important diseases of
protozoa and fungi (Lightner and Redman 1998). Acute hepatopancreatic necrosis disease
(AHPND), formerly known as early mortality syndrome (EMS), is a bacterial shrimp disease
due to the action of PirABvp toxin secreted by Vibrio parahaemolyticus. The outbreak of
AHPND has plagued the aquaculture industry with substantial economic losses since 2009
(Lai et al. 2015). AHPND can also be caused by other strains of Vibrios such as Vibrio
harveyi,Vibrio punensis,Vibrio owensii,Vibrio campbellii,andVibrio alginolyticus which
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possess pVA1-type plasmid where the genes encoding the toxin are located (Dong et al. 2019).
V. alginolyticus is an opportunistic pathogen of shrimp known to cause mortalities under
poor environmental conditions (Liu and Chen 2004). Vibrio harveyi, which causes lumi-
nous vibriosis, is another etiological agent for mass mortalities in Penaeid shrimp
(Karunasagar et al. 1994). White Spot Syndrome Virus (WSSV) and Infectious
Myonecrosis Virus (IMNV) are the other two most important viral pathogens affecting
shrimp aquaculture. WSSV is highly virulent and causes total mortality in shrimp within 3
to10 days after the appearance of clinical symptoms (Sánchez-Martínez et al. 2007). In
contrast, IMNV is less threatening as mortalities are reported under unfavorable physico-
chemical conditions (Prasad et al. 2017).
Shrimp larvae are particularly susceptible to mycosis caused by fungi such as
Saprolegnia parasitica and Achlya flagellate. The fungi enter larvae through antenna,
eyestalks and invade body tissues, ultimately leading to death (Hubschman and Schmitt
1969). Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that retards shrimp
growth (Rajendran et al. 2016) and often causes white feces syndrome (WFS) in cultured
shrimp (Tangprasittipap et al. 2013).
In comparison to wild-caught shrimp, antibiotic resistance is much more pronounced in
farmed shrimp, suggesting the unsystematic usage of antibiotics in shrimp farms for increasing
the yield of production (Boinapally and Jiang 2007). The choice of antimicrobials used in
farming practices is often based on the knowledge of shrimp farmers or chemical suppliers and
not from veterinary services (Chi et al. 2017; Ali et al. 2016). This is the main reason why
indiscriminate use of chemotherapeutic agents prevails in the aquaculture sector.
Previous studies have reported shrimp pathogens belonging to the Vibrio genera showing
resistance to antibiotics such as tetracycline and β-lactam antibiotics, from aquaculture settings
(Nakayama et al. 2006;Meloetal.2011; Albuquerque Costa et al. 2015). Karunasagar et al.
(1994) reported mass mortalities of shrimp larvae due to antibiotic-resistant Vibrio infections,
which suggests that antibiotic-resistant pathogens can be highly devastating. The source of
antibiotic-resistant strains in shrimp farms could be the seawater collected for rearing. Draining
antibiotics and other chemicals containing effluents into the sea may have contributed to the
emergence of antibiotic-resistant Vibrios in marine ecosystems (Srinivasan and Ramasamy
2009).
Plasmids in Vibrio have a considerable role in disseminating antibiotic-resistant genes to
other bacteria, making it a huge problem from the environmental perspective (Sengupta and
Austin 2011). It was experimentally demonstrated that the Vibrio strain HL60 could transfer
cephalothin resistant genes present in the plasmid to Escherichia coli (Molina-Aja et al. 2002).
Kitiyodom et al. (2010) found that integrons could transmit antibiotic resistance in multi-drug
resistant Vibrios isolated from shrimp farms of Thailand. Presence of antibiotic-resistant genes
and their dissemination in aquaculture sites is a clear indication of a threat to food safety (Su
et al. 2017). Antibiotic-resistant forms of food-borne pathogens such as Salmonella (Banerjee
et al. 2012;Carvalhoetal.2013)a
ndBacillus spp. (Le et al. 2005) have also been reported to
be present in farmed shrimp, which makes antibiotic resistance in shrimp aquaculture a
legitimate issue to be resolved. The countries at a greater risk of climate change will likely
face a higher incidence of antibiotic resistance in the future, thus extending its threat not just to
aquatic life but also to human health (Reverter et al. 2020). Therefore, it is vital to develop
sustainable alternatives to antibiotics in shrimp aquaculture as a disease management strategy.
An overview of the pros and cons of the alternative strategies discussed throughout the review
is represented in Table 1.
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Nanomaterials and their versatile applications in shrimp aquaculture
Nanoparticles are particles ranging from 1 to 100 nm with unique properties attributed to their
size (Bowman 2017). Metallic salts are reduced to form nanoparticles with thehelp of reducing
agents (Van Hyning et al. 2001), whereas polymeric nanoparticle synthesis is carried out by
polymerizing monomers by various methods (Nasir et al. 2015). Nanotechnology is applied in
the field of biology for the development of drug delivery systems, bio-imaging, and gene
delivery. It offers a variety of applications in aquaculture as carriers in vaccine delivery,
diagnostic tools, antimicrobial agents, and anti-biofilm agents (Fig. 1).
Vaccine delivery
Vaccination is practiced in aquaculture as a prophylactic measure to improve host immunity
against infections. Several researchers have focused on developing oral DNA vaccines for
shrimp using carriers like PLGA (poly-lactide-coglycolide acid) and chitosan for viral and
bacterial diseases. The carriers are now widely used because they are biodegradable, water-
Table 1 A brief summary of advantages and disadvantages of different strategies employed as an alternative to
antibiotic therapy
Antimicrobial
agent
Pros Cons
Nanomaterials •Versatile applications in infection therapy as
antimicrobial agents, antibiofilm agent (Moges
et al. 2020) and vaccine components
(Rajeshkumar et al. 2009).
•Environmental toxicities associated
with the use of nanoparticles (Zoroddu
et al. 2014)
•Broad spectrum antimicrobial activity against
pathogens (Camacho-Jiménez et al. 2020).
Phages •Effective tool for the delivery of genes and
antigens (Solís-Lucero et al. 2016;Dyetal.
2018).
•Stabilization of phage formulation for
therapy is cumbersome task to
accomplish (Ssekatawa et al. 2021).
•Availability of multiple routes in administration
of phages (Nakai et al. 1999; Nakai and Park
2002).
•Difficulty in isolating phages that do
not harbor genes conferring antibiotic
resistance and virulence (Ssekatawa
et al. 2021).
Biofilm-based
vaccines
•Improved immune response can be achieved
without the use of adjuvants (Vinay et al.
2016).
•Possible immune tolerance due to high
or low dosing (Vinay et al. 2016).
Phytobiotics •Multiple health benefits including improved
growth and relief from oxidation stress
(Elumalai et al. 2020).
•Possibility in the development of
antibiotic resistance when single
active ingredient is used (Gupta and
Birdi 2017).
•Short duration outcomes were found to be as
effective as long-term ones (Reverter et al.
2021)
Algal extracts •Enhances growth and performance of shrimp
along with antimicrobial activity (Lee et al.
2020).
•Disparities in bioactive molecules
obtained depending on the extraction
method employed (Klongklaew et al.
2020)
Probiotics,
Prebiotics
and
Synbiotics
•In addition to immunomodulation and
antimicrobial activity, they can sustain intestinal
health (Amenyogbe et al. 2020).
•Complications related to the
development of species-specific
probiotics, prebiotics and synbiotics
(Amenyogbe et al. 2020).
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soluble, and non-toxic (Chalamcherla 2015). Chitosan/DNA construct (pVp28) nanoparticles
administered orally in P. monodon showed improved immunity and enabled protection against
WSSV (Rajeshkumar et al. 2009). Chitosan nanoparticles loaded with the outer membrane
protein K (ompK) gene of V. parahemolyticus stimulated an immune response against the
pathogen in Acanthopagrus schlegelii (Li et al. 2013); however, its efficacy in shrimp is
unknown. Liposome-based vaccines have also been experimented in shrimp aquaculture as
they are considered safe because of their compositional similarity to that of cell membranes.
Liposome-based nanoparticles have an additional advantage in protecting the antigenic com-
ponent from hydrolytic enzymes and pH changes, ensuring effective antigen delivery in the
host. Liposome-based recombinant VP28 vaccine when administered orally for 7 days against
WSSV showed 78.9% survival rates in Marsupenaeus japonicus (Mavichak et al. 2010).
Experimental studies regarding the development of spike protein-based nanoparticles,
dendrimers, and carbon nanotubes have not progressed much in aquaculture compared to
human medicine. The vaccination strategies presently followed in aquaculture can be im-
proved considerably with the help of these nanoparticles as they serve as a means of mass
vaccinations without the burden of injections.
Diagnostic tools
Nanoparticles have become an integral part of diagnostic methods employed for the detection
of human and animal pathogens. Gold nanoparticles have gained a lot of attention in
diagnosing shrimp diseases of viral, bacterial, and protozoal origin. Notomi et al. (2000)
described loop-mediated isothermal amplification in which a target DNA is amplified in
isothermal conditions efficiently even in the presence of undesirable DNA fragments. This
Fig. 1 Applications of nanomaterials in shrimp aquaculture. Multiple applications of nanomaterials such as
disease diagnosis, anti-microbial agents, anti-biofilm agents, and delivery of vaccines to shrimp have been
illustrated
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detection method requires agarose gel electrophoresis and excludes a hybridization step that
would help understand DNA structure’s nature.
Gold nanoparticles can be bound to alkyl thiol-terminated oligonucleotides as they are
highly stable in saline conditions allowing hybridization with a complementary strand of DNA
(Arunrut et al. 2016). Nanoparticles have surface plasmon resonance, which depends on the
distance between the particles. Thus, when nanoparticles clump together, their surface plasmon
resonance absorption band changes in the visible region accompanied by a color change from
red to purple-blue. This property is utilized for colorimetric detection of successful hybridiza-
tion visible to the naked eye (Arunrut et al. 2016). This method also saves time as PCR or gel
electrophoresis is not required. Gold nanoparticles were found to improve the sensitivity of
immune assays and allowed detection of small quantities of anti-sera. An immune dot-blot
assay for detecting WSSV in shrimp was developed by raising a secondary antibody against
the anti-sera of WSSV in hemolymph (Thiruppathiraja et al. 2011). These secondary antibod-
ies were conjugated with the enzyme alkaline phosphate, linked to gold nanoparticles. The
newly devised method had 91.6% efficiency compared to the conventional immune dot-blot
assay and detected even 1ng/mL concentration of antibodies against WSSV (Thiruppathiraja
et al. 2011).
Gold nanoparticles were used to develop the Reverse Transcription-Loop-mediated iso-
thermal amplification/visual colorogenic nano-gold hybridization probe assay to detect the
Yellowhead virus in shrimp. In this study, the gold nanoparticles were used for probing DNA
strands complementary to viral DNA (Jaroenram et al. 2012). A modified Loop-mediated
isothermal amplification method with the incorporation of gold nanoparticles against
microsporidian E. hepatopenaei could detect even 0.02 fg in the sample (Suebsing et al.
2013). Similarly, gold nanoparticles were used to improve loop-mediated isothermal amplifi-
cation in detecting shrimp pathogens, IMNV, and V. parahaemolyticus (VPAHPND)(Arunrut
et al. 2013; Arunrut et al. 2016). Exploring other nanomaterials with high-quality sensor
properties will aid the development of nano-based biosensors for the detection of infectious
diseases in aquaculture.
Antimicrobial agents
Nanoparticles have been used as antimicrobial agents in the areas of medicine, food, and
agriculture. Nanoparticles can be fed to shrimp by mixing it with the feed or applied in the
water used for rearing (Luis et al. 2019). These are the common practices for nanomaterial
application, but innovations have been introduced recently. Sarkheil et al. (2016)
immobilized silver nanoparticles (AgNPs) on silica beads in a water filter column capable
of killing luminous Vibrio sp. Persian1. Filtered seawater used for rearing the post larval
stage of P. vannamei improved not only their survival rate but also their growth
performance.
The nanoparticle size is proved to be one of the key factors that influence antimicrobial
nature. Smaller nanoparticles have better antimicrobial activity, and this property is attributed
to the large surface area to volume ratio in smaller particles (Ramamoorthy et al. 2013). Gold
nanoparticles (AuNPs) of size 18 nm exhibited immunostimulation and reduced AHPND
associated histopathological damages in shrimp without exerting any signs of toxicity upon
single oral administration (Tello-Olea et al. 2019).
Among the metal nanoparticles, AgNPs are more commonly used in shrimp aquaculture as
it has been highly studied and frequently reported due to its performance as a robust
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antimicrobial agent (Meneses-Márquez et al. 2019). Besides its antimicrobial activity, AgNPs
have been assessed to find any possible toxicities associated with their use in shrimp. AgNPs
administered intramuscularly to juvenile whiteleg shrimp P. vannamei, at a concentration
below 20 ng/μL showed no signs of behavioral changes or mortality after 96 h. Also, the
routine oxygen consumption rate and total hemocyte counts in shrimpwere found to be normal
after administering AgNPs, suggesting that shrimp were not under stress (Juárez-Moreno et al.
2017).
AgNPs derived from the leaf extract of Camellia sinensis after long-term treatment in
Feneropenaeus indicus infected with V. harveyi showed a 71% decrease in mortality caused
by the pathogen (Vaseeharan et al. 2010). AgNPs synthesized using coastal plant Prosopis
chilensis when fed for 30 days in P. monodon challenged with Vibrio cholerae,V. harveyi,and
V. parahaemolyticus had a higher hemocyte count and phenoloxidase activity suggesting
immunomodulatory effects (Kandasamy et al. 2013). Green AgNPs synthesized using tea
and neem leaf extract proved to treat shrimp infected with necrotizing hepatopancreatitis
bacterium (NHP-B), a rickettsial pathogen (Acedo-Valdez et al. 2017). Alvarez-Cirerol et al.
(2019) synthesized AgNPs using the plant extract of Rumex hymenosepalus and found that the
nanoparticle inhibited V. parahemolyticus and increased the survival of post-larval shrimp.
AgNPs synthesized using seagrass Cymodocea serrulate showed restoration of
hepatopancreatic function when fed to Penaeus monodon infected with V. parahaemolyticus
(Rathnakumari et al. 2018).Maldonado-Muñiz et al. (2020) used extracts of seaweed Ulva
clathrate for AgNP synthesis and found that treatments with nanoparticles showed no lethality
after feeding the shrimp for 7 days. AgNPs synthesized using Spirulina extract reduced the
virulence of V. parahemolyticus and enhanced survival of Artemia nauplii challenged with the
pathogen (Palanisamy et al. 2017). Antiviral activity of commercially available AgNP called
Argovit, against WSSV, was reported by Juárez-Moreno et al. (2017) and Romo-Quiñonez
et al. (2020).
The majority of studies have reported metallic nanoparticles, and only a few have reported
the use of polymeric nanoparticles as an antimicrobial agent in aquaculture. Chitosan nano-
particles upon dietary administration for 12 days improved immunity against WSSV in
crayfish and inhibited virus replication (Sun et al. 2016). Metallic nanoparticles are non-
biodegradable and promote biomagnification, whereas polymeric nanoparticles are less toxic
to the biological system and are environmentally benign (Moges et al. 2020). Given that
polymeric nanoparticles enable the sustained release of drugs, the application of antimicrobial
agents loaded in nanoparticles can open new horizons in shrimp disease management.
Anti-biofilm agents
Quorum sensing is a cell-to-cell communication system observed in bacteria that controls the
expression of virulent genes and biofilm formation (Nealson et al. 1970). Aquaculture
pathogens such as V. harveyi,V. parahaemolyticus, Vibrio anguillarum, and Vibrio vulnificus
are capable of quorum sensing (Defoirdt et al. 2004). Biofilm forming pathogens cause
persistent infections and are recalcitrant to antibiotic therapy (Karunasagar et al. 1996). This
calls for suitable alternatives to inhibit biofilm infections of aquaculture pathogens.
AgNPs synthesized using Gelidiella acerosa, red algae, inhibited biofilm formation,
production of extracellular polysaccharide (EPS), swimming, and swarming motility without
affecting the growth of V. vulnificus and V. parahaemolyticus (Satish et al. 2017). Copper
nanoparticles were found to retard EPS production and cell surface hydrophobicity (CSH) and
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hence controlled the biofilm formation by V. alginolyticus, V. parahaemolyticus, and
A. hydrophila (Chari et al. 2017) (Table 2).
These studies have reported the efficacy of nanoparticles under in vitro conditions against
the shrimp pathogens. Lack of in vivo studies limits the knowledge on the virtue of
nanoparticle-based biofilm treatment in aquatic animals.
Despite their attractive properties and vast applications in aquaculture, several reports
support the toxicity associated with the use of metallic nanoparticles in the environment
(Zoroddu et al. 2014). Studies conducted so far have analyzed the advantage of nanoparticles
with short-term exposure. Whether or not these nanoparticles have a detrimental effect on
shrimp or develop antimicrobial resistance to nanoparticles after a long time of exposure needs
to be addressed. Also, safety assessment of nanoparticle treated shrimp for human consump-
tion have to be studied before use. Green and biological synthesis of nanomaterials is
considered safe and environmentally benign (Singh et al. 2018). More application studies in
aquaculture utilizing green and biologically synthesized nanoparticles will allow their success-
ful implementation in the future.
Biofilms in shrimp aquaculture: the positive side
As mentioned earlier, biofilms of pathogens can cause persistent infections. Bacterial
biofilms as a source of immunostimulants for fishes have emerged to be a successful
strategy since they can be used directly without encapsulation or adjuvants for delivery
(Vinay et al. 2016). The expression of proteins in biofilm cells and planktonic cells are
dissimilar; therefore, cell vaccines with new immunogens can be developed using biofilms
(Mamun et al. 2019). In addition, oral immunostimulation is a more fiscal and rational
strategy for aquaculture practices (Sharma et al. 2010). Biofilm-based immunostimulants
are prepared by growing biofilms of pathogens on chitin flakes, which are inactivated either
by heat or formalin and are then mixed with feed using a binder (Mamun et al. 2019;
Sharma et al. 2010).
The supplementation of inactivated V. alginolyticus biofilms on chitin flakes in the feed
showed antimicrobial effects, enhanced phenoloxidase activity, and rise in total haemocyte
count, thus stimulating immunity of shrimp (Sharma et al. 2010). P. monodon fed with biofilm
cells of V. alginolyticus at a dose of 109CFU/g of shrimp for 14 days displayed resistance to
V. alginolyticus and WSSV infections. Similarly, inactivated biofilms of V. harveyi supple-
mented in shrimp feed for immunostimulation of P. vannamei showed enhanced lysozyme
activity, increased production of penaeidin, crustin and antimicrobial peptides (Vinay et al.
2019). Heat-inactivated cells of A. hydrophila were developed as novel antigens for oral
vaccination in fishes (Mamun et al. 2019). This can be extended in whiteleg shrimp for
immunostimulation since A. hydrophila is reported as an emerging bacterial pathogen (Zhou
et al. 2019).
Beneficial role of bacteriophages in shrimp aquaculture
Bacteriophages are viruses that can integrate into the bacterial DNA and induce the lysis of the
bacterialcells. The tail and tail fibers of bacteriophages help in the adherence of the virus to the
host receptors present on the outer surface of bacteria, following which the viral particles inject
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Table 2 Antibacterial, antiviral, and anti-biofilm activity of nanoparticles against shrimp pathogens
Pathogen Nanoparticle Activity Mode of synthesis Concentration Size and shape of the nanoparticle References
V. parahaemolyticus AuNP Antibacterial
activity
Chemical (Turkevich method) 2 μg/g Ovoid shape, 18.57 ± 4.37 nm Tello-Olea et al.
(2019)
V. parahaemolyticus AgNP Biological (Spirulina extract) 5 μg/mL spherical shape,
2- 18nm.
Palanisamy et al.
(2017)
V. harveyi AgNP Commercial (Nanocid) 391 μg/mL 16.62 nm Bahabadi et al. (2017)
V. parahaemolyticus AgNP Biological (U. clathrate
extract)
3.2 μg/mL spherical shape, 5 to 20 nm. Maldonado-Muñiz
et al. (2020)
V. parahaemolyticus AgNP Biological (R. hymenosepalus
extract)
25 μg/mL quasi-spherical geometry, size 2 and
10nm.
Alvarez-Cirerol et al.
(2019)
Vibrio harveyi AgNP Biological (C. sinensis extract) 10 μg/mL - Vaseeharan et al.
(2010)
V. cholerae, V. harveyi and V.
parahaemolyticus
AgNP Biological (P. chilensis
extract)
- spherical shape, 5 to 25 nm Kandasamy et al.
(2013)
Necrotizing hepatopancreatitis
bacterium (NHP-B)
AgNP Biological (Azadirachta indica
aextract)
35 μg polyhedral, semi-hemispherical and
flattened shapes,
5to45nm.
Acedo-Valdez et al.
(2017)
WSSV AgNP Antiviral
activity
Commercial (Argovit) 5 and 20
ng/mL
spheroid shape ,35 ± 15 nm diameter Juárez-Moreno et al.
(2017)
WSSV AgNP Commercial (Argovit) 1,000 μg/g - Romo-Quiñonez et al.
(2020)
WSSV Chitosan NP Physicochemical (Ultrafine
milling method)
10 mg/g spherical shape, size ranging from 20
to 1000 nm
Sun et al. (2016)
V. vulnificus and V. parahaemolyticus AgNP Anti-biofilm
activity
Biological ( G. acerosa
extract)
100 μg/mL spherical shape, size ranging from 20
to 50 nm
Satish et al. (2017)
V. alginolyticus,V. parahaemolyticus
and A. hydrophila
CuNP Chemical (One-pot synthesis
method)
100 ng/mL Spherical shape with average size of
55nm
Chari et al. (2017)
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their genetic material into the bacterial host (penetration) after which the phage undertakes the
host machinery for its replication (Weinbauer 2004). Once the viral components are synthe-
sized, they are assembled to form mature phage particles that are eventually released out of the
cell by lysis followed by a repetition of the cycle (Weinbauer 2004). The principal advantage
of using phages for treatment is their abundance, ease of multiplication by regulating bacterial
growth, environment-friendly characteristics, and host-specificity as they will not interfere
with gut microbiota and therefore show minimal side effects when compared to antibiotic
therapy (Oliveira et al. 2012). Phages have been used to control bacterial diseases, and some
are genetically engineered for antiviral therapies.
Phages against bacterial infections in shrimp
Phage therapy is carried out by administering the phages through injection, dietary adminis-
tration, and incorporation in rearing systems (Nakai et al. 1999; Nakai and Park 2002). But
dietary administration is desirable as it helps treat large populations and prevents infections in
the intestine (Nakai 2010).
Nikapitiya et al. (2020) reported that live Artemia nauplii supplemented with Edwardsiella
tarda phage (ETP-1) when fed to zebrafish did not impart any adverse immune reaction
suggesting that this method of phage delivery can be inculcated for the treatment of bacterial
diseases in aquaculture. Phage therapy was found to be successful in treatment at all stages of
infection caused by V. parahaemolyticus in P. vannamei larvae (Lomelí-Ortega and Martínez-
Díaz 2014). In vitro studies showed that the phage pVp-1 was capable of lysing acute
hepatopancreatic necrosis disease (AHPND) causing strains of V. parahaemolyticus (Jun
et al. 2016) and later was found to have prophylactic as well as therapeutic potential in shrimp
(Jun et al. 2018). Phage cocktails, which are a combination of phages, have been found to act
synergistically than using single phages. Mateus et al. (2014) studied three phages VP-1, VP-2,
and VP-3 isolated from marine water against V. parahaemolyticus and found their combination
to be more effective. Makarov et al. (2019) combined T2A2 and VH5e bacteriophages for
effective lysing of AHPND causing V. parahaemolyticus strains. Ma et al. (2019) developed a
phage cocktail consisting of five novel phages belonging to the family of Siphoviridae, which
inhibited the growth of Vibrio sp. Va-F3 and survival rate of infected shrimp. Kalatzis et al.
(2016) administered a suspension of phages φSt2 and φGrn1 to brine shrimp Artemia salina
infected with V. alginolyticus strain V1 and observed that the load of bacteria was reduced by
93% within 4 hrs. Phages have also been documented for anti-biofilm activities against shrimp
pathogens. Prada-Peñaranda et al. (2018) used a phage preparation FBL1, targeting biofilmsof
Bacillus licheniformis strain 52 known to cause high mortalities in juvenile and adult
P. vannamei. The phage preparation reduced the biofilms of B. licheniformis to 44.77%
(Prada-Peñaranda et al. 2018).
Apart from thedirect use of phagecocktails for bacterial lysis, endolysin genes from phages
have been cloned and expressed for therapeutical application in aquaculture. The phage-based
endolysin (Vplys60) expressed in yeast Pichia pastoris showed in vitro antibacterial activity
against V. parahaemolyticus at 75 μg/mL and reduced bacterial load in Artemia franciscana
(Srinivasan et al. 2020). Thammatinna et al. (2020) identified a vibrio phage named Seahorse,
interfering with the protein machinery of AHPND causing V. parahaemolyticus, indicating the
presence of an antimicrobial compound encoded by the phage. These studies point out
different possibilities of using phages in shrimp aquaculture in controlling bacterial diseases
(Table 3).
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Table 3 Various applications of bacteriophages in shrimp aquaculture
Name of the phage/phage product In vivo host Titre of phage
used/phage prod-
uct
Targeted pathogen Key findings References
ETP-1 Zebrafish 1011 PFU/mL - No phages persisted in spleen and liver after 7 days of feeding
with artemia enriched phages.
Nikapitiya
et al.
(2020)
VP-1, VP-2, and VP-3 - 107PFU mL V. parahaemolyticus Phages were viable for upto 7 months Mateus
et al.
(2014)
Bacterial inhibition upto 4.2 log within 6 h incubation
pVp-1 P. vannamei 1.5 × 108
PFU/shrimp
V. parahaemolyticus Combination of prophylactic and therapeutic administration of
phage by oral route caused highest reduction in mortality
Jun et al.
(2016)
T2A2 and VH5e A. franciscana MOI = 1 V. parahaemolyticus
CVP2
Phage cocktail was active against bacterial suspension of 6 × 106
and 6 × 107CFU/ml tested in vivo
Makarov
et al.
(2019)
Phage cocktail of VspDsh-1, ValLY-3,
ValSw-1, VpaJT-1, and VspSw-1
P. vannamei 2×10
7PFU/ml Vibrio sp. Va-F3 Survival rate of shrimp was 91.4% after 7 days of treatment Ma et al.
(2019)Phage cocktail exhibited inhibitory activity against
phage-resistant Va-F3 variants
φSt2 and φGrn1 A. salina MOI = 100 V. alginolyticus Vibrio load in Artemia was reduced to 5.3 x 103±3.1 x 103
CFU/mL after 4 h of phage treatment
Kalatzis
et al.
(2016)
Peptide 2E6 Freshwater
crayfish
50 μL WSSV Mortality was reduced to 33.38 % with an increase in median
lethal time (LT50) for 20 days
Yi et al.
(2003)
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The resistance of bacteria to phages occurs when the virus cannot adhere to the cell
membrane due to loss of receptors (Oliveira et al. 2012). In addition to this, restriction
enzymes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sys-
tems in bacteria also render resistance to phage infection (Hyman and Abedon 2010). A
study conducted by Laanto et al. (2020) found that myophage FCV-1-resistant clones of
Flavobacterium columnare emerged within 1 day of co-culture. But after a week, phage
isolate capable of infecting more than half of the previous phage-resistant clones were
identified (Laanto et al. 2020). Hence, trained phages that have naturally co-evolved to
infect phage resistant clones can be used while preparing phage suspension for treatment
purposes.
Generally, in phage therapy, only lytic phages are preferred as temperate phages can
lysogenise bacterial cells and transfer virulent genes/ antibiotic-resistant genes via horizontal
gene transfer (Oliveira et al. 2015). But some reports have confirmed the presence of
horizontal gene transfer in lytic phages via transformation and transduction. Two natural lytic
phages were found to transmit transformable plasmid DNA bearing antibiotic-resistant genes
from Escherichia coli to Bacillus sp. (Keen et al. 2017), and E. coli phages from urban
wastewater was found to transduce β-Lactam resistance genes (Gunathilaka et al. 2017).
Besides this, the existence of virulent mutants of temperate phages in nature should not be
ignored as they can revert to lysogeny, complicating the treatment (Krylov 2001). These
findings indicate the possible hazards of using phages for the treatment of antibiotic-resistant
infections.
Phage display technology to combat viral diseases in shrimp aquaculture
WSSV is a major concern for shrimp aquaculture globally and has caused an estimated loss of
seven billion dollars in shrimp aquaculture industries located in Asia and America in the 1990s
(Arulmoorthy et al. 2020). Yi et al. (2003) constructed a 10-mer phage display peptide library
and found that the peptide 2E6, with VAVNNSY critical motif, reduced pathological signs of
WSSV infection in freshwater crayfish. Phage display technology for detecting viral pathogens
is cost-effective because it does not demand any antigen purification steps, and neutralizing
scFv antibody synthesis is carried out in bacterial hosts (Yuan et al. 2006). Yuan et al. (2006)
reported a neutralizing antibody from the anti-WSSV single-chain fragment variable region
(scFv) antibody phage display library, which could be a potential tool for WSSV detection and
treatment. Phage display technology has also been implemented in vaccination technique for
the delivery of antigens. Solís-Lucero et al. (2016) constructed a recombinant phage that
expressed VP28 protein for vaccinating shrimp against WSSV infection. Phage display
technology can be extended for other infectious viral diseases in shrimpto identify neutralizing
antibodies and potential antigens for prophylaxis and disease diagnosis.
Phytobiotics for shrimp disease management
Phytobiotics are plant extracts that generally contain bioactive compounds like phenolics,
tannins, flavonoids, and essential oils with antimicrobial and immunomodulating activity
(Chakraborty and Hancz 2011). Phytobiotics have less tendency to induce resistance in
pathogens, which can be attributed to various molecules present in them and are cost-
effective and eco-friendly than synthetic compounds like antibiotics (Logambal et al. 2000;
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Blumenthal 2000; Olusola et al. 2013). The oral route of administration is being widely
practiced since it is found to be effective and easy (Yoshida et al. 1995).
Phytobiotics as an immunomodulating agent
Several pieces of research have reported the immunomodulating effects of plant extracts
in shrimp. Leaf extracts of mangrove plant Xylocarpus granatum improved the survival
rate of shrimp post V. harveyi infection by increasing the population of haemocytes such
as hyaline cells, semi-granular, and granular in shrimp (Saptiani et al. 2020a). The
powdered form of bulbs of the medicinal plant Eleutherine bulbosa administered in
shrimp feed improved immune genes expression and restricted V. parahaemolyticus
infection (Munaeni et al. 2020). Lee et al. (2020) observed that injection of Theobroma
cacao pod husks in shrimp resulted in an enhanced expression of immune genes like toll-
like receptors (TLR- 1), signal transducer and activator of transcription (STAT), and
crustin in shrimp, concluding its immunomodulating effect on the innate immune system
of shrimp.
Individual components from plant extracts have also been employed as immunomodulating
agents for shrimp. α-Phellandrene, a compound present in essential oils of herbs upregulated
mRNA levels of immune genes lipopolysaccharide- and β-1,3-glucan-binding protein,
prophenoloxidase, and peroxinectin after V. alginolyticus challenge (Wu et al. 2019). These
studies collectively demonstrate that plant extracts could be used as immune stimulators in
shrimp farming.
Phytobiotics as antimicrobial agents
Phytobiotics have been extensively studied in shrimp aquaculture as they harbor compounds
that can interfere with the bacterial cell wall and viral mRNA synthesis (Jana et al. 2018). The
ethanolic extract of the plant Cynodon dactylon contained antiviral compounds like epicate-
chin, p-coumaric acid, catechin, vanillic acid, syringic acid, and quercetin, which rendered
protection against WSSV in shrimp upon intramuscular injection of the extract (Howlader
et al. 2020). Olea europaea leaf extracts, when orally administered to shrimp juveniles,
improved the survival rate by 65% towards WSSV infection (Gholamhosseini et al. 2020).
Ascorbic acid monophosphate and Phytocee™, a commercial phytogenic feed comprising of
the extracts of Emblica officinalis, Ocimum sanctum, and Withania somnifera, conjointly
increased the survival rate of shrimp challenged with WSSV (Selvam et al. 2020). Enrichment
of chitosan and mangrove flour as windu shrimp feed was also found to reduce the mortality
associated with WSSV in shrimp (Manik et al. 2020). Cahyadi et al. (2020)fortifiedA. salina
using the fruit extract of Sonneratia alba and orally administered the bio-enriched Artemia to
inhibit V. harveyi infection in shrimp.
Apart from antibacterial and anti-viral activity, certain phytochemicals have shown prom-
ising anti-fungal and anti-parasitic activities against shrimp pathogens. S. alba leaf extract was
capable of preventing Saprolegnia sp. and V. harveyi infection in tiger shrimp when admin-
istered in the rearing system (Saptiani et al. 2020b). Plant products like ginger paste (Zingiber
officinale), lemon juice (Citrus limon), garlic homogenate (Allium sativum), jaggery powder
(Borassus flabellifer), black gram flour (Vigna mungo) is mixed with shrimp feed by shrimp
farmers of South India to curb E. hepatopenaei infections effectively (Palanikumar et al. 2020)
(Table 4).
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Table 4 Applications of phytobiotics in shrimp aquaculture
Phytobiotic Purpose Pathogen In vivo host Dosage and period of administration References
E. bulbosa bulb powder Immunostimulants V. parahaemolyticus P. vannamei 12.5 g/Kg of shrimp for 30 days Munaeni et al.
(2020)
Hot-water extract T. cacao pod
husk extract
V. alginolyticus P. vannamei 40 μg/ shrimp injected once Lee et al. (2020)
Distilled water extract of
X.granatum leaf extract
V. harveyi P. monodon 1.250 ppm Saptiani et al.
(2020a)
α-Phellandrene V. alginolyticus P. vannamei 8 and 12 μg/Kg of shrimp for 72 h Wu et al. (2019)
Ethanolic extract of C. dactylon Antiviral WSSV P. vannamei 150 mg/Kg body weight injected once Howlader et al.
(2020)
Methanolic leaf extract of
O. europaea
WSSV P. vannamei 200 mg/Kg of the shrimp biomass for 7 days Gholamhosseini
et al. (2020)
Ascorbic acid monophosphate and
Phytocee™
WSSV P. vannamei 500 g/ton feed of ascorbic acid monophosphate and 1000 g/ton
feed of Phytocee™fed for 42 days
Selvam et al.
(2020)
Chitosan and mangrove flour WSSV P. monodon 300 g of Chitosan and mangrove flour per 1 Kg of commercial
feed
Manik et al. (2020)
Saline water extract of S. alba Anti-fungal Saprolegnia sp. P. monodon 1250 mg/L added once in the rearing system Saptiani et al.
(2020b)Antibacterial V. harveyi
Ethanolic fruit extract of S. alba
enriched in A. salina
V. harveyi P. monodon 20 ppm of extract for 10 days Cahyadi et al.
(2020)
Z. officinale paste Anti-parasitic E. hepatopenaei P. vannamei 20 g /Kg of feed Palanikumar et al.
(2020)
C. limon juice 50 mL/Kg of feed
A. sativum homogenate 20 g/ Kg of feed
B. flabellifer powder 30 ml of jaggery syrup (1 Kg/L) per 1 Kg of feed
V. mungo flour 50g/Kgoffeed
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Most of these studies have focused on assessing the antimicrobial activity of the plant
extract as a whole, rather than identifying the key molecule possessing antimicrobial property.
It is necessary to identify the active components from the extract to calculate the dosage and
toxicity level and understand their mechanism of action before large scale applications.
Therapeutic applications of marine algal extracts for shrimp disease
management
Marine algae are known to produce bioactive compounds, and compared to antibiotics, these
compounds are safe to the environment and have good antimicrobial properties, due to which
they can be used in shrimp aquaculture (Dashtiannasab et al. 2016). Algal extracts can be
administered to shrimp by injection, immersion, and feed supplement (Huang et al. 2006;Yeh
et al. 2006). Hot-water extract of the seaweed Sargassum duplicatum at a concentration below
10 μg/g, when injected into P. vannamei, enhanced immune response against
V. parahemolyticus infection (Yeh et al. 2006). Selvin et al. (2011) formulated a medicated
diet using the secondary metabolites of marine algae, Ulva fasciata which was highly effective
in controlling shrimp bacterial infections at a dose of 1 g/kg of shrimp. Apart from bacterial
infections, seaweed extracts have also been studied against viral infections. The extract of the
algae, Sargassum hemiphyllum var. chinense when administered to white shrimp exhibited
improved resistance to WSSV infections (Huynh et al. 2011).
Metabolites derived from marine algae are incorporated in shrimp feeds to improve the
resistance of shrimp to infections. The polysaccharide fucoidan from the algae, Sargassum
wightii when supplemented at 0.3% in the feed of P. monodon for 45 days, showed a decrease
in mortality associated with WSSV infection (Immanuel et al. 2012). An antiviral compound,
2-(2-hydroxyphenoxy)-1-phenylethanol from Enteromorpha flexuosa at 400 mg/kg when fed
to F. indicus provided resistance to WSSV infection due to augmented immunity (Velmurugan
et al. 2015). Dietary administration of polysaccharide extract of the seaweed Sargassum
fusiforme in juvenile shrimp, Fenneropenaeus chinensis enhanced resistance against vibriosis
and improved its immunity (Huang et al. 2006). Dietary administration of the protein extract of
red seaweed Gracilaria fisheri at 100 μg/ mL in whiteleg shrimp exhibited better survival and
had normal histological features of hepatopancreas when challenged with V. parahaemolyticus
(Boonsri et al. 2017).
Marine algae are an excellent source of quorum quenching compounds that can reduce the
biofilm formation of bacterial pathogens of shrimp. Ethanolic extract and furanone from
G. fisheri inhibited biofilms of V. parahaemolyticus and V. harveyi, respectively, at sub-
MIC concentrations and reduced mortality of shrimp that received these compounds through
diet (Karnjana et al. 2019).
The cost involved in extraction methods to obtain algal compounds is high (Ibañez et al.
2012); therefore, new techniques should be developed to perform the extraction in a cost
convenient manner. A study conducted by Manilal et al. (2009) showed that the antimi-
crobial property of extract of Asparagopsis taxiformis varied seasonally and highest
activity against shrimp pathogens, was recorded in the months of December and January.
Thus, the compositional analysis of algal extracts with respect to seasonal variations has to
be considered while developing antimicrobial compounds for aquaculture. Further analysis
and characterization of bioactive compounds from the algal source are required to develop
various formulations.
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Microbial interventions in shrimp aquaculture
Probiotics are mono or mixed culture of live microorganisms used to improve the properties
of the indigenous microflora (Havenaar and Huis In’tVeld1992), boost shrimp innate
immunity and work antagonistically towards the pathogens by secreting antimicrobial
compounds (Hai and Fotedar 2010). The probiotic microorganisms provide digestive
enzymes to the shrimp and compete for nutrients in the environment, thus keeping the
pathogen population in control (Irianto and Austin 2002). Probiotics can be administered
by immersing shrimp in probiotic suspension or orally by mixing them with feed (Itami
et al. 1998; Sakai 1999). Oral administration is much more effective in treatment because it
targets shrimp of all stages (Sakai 1999), while the immersion method is effective in the
juvenile stage (Itami et al. 1998).
Prebiotics are dietary components that support the sustenance of beneficial microorganisms
in the gut (Hai and Fotedar 2009). Prebiotics are mostly indigestible carbohydrates, which
could be a monosaccharide, oligosaccharide, or polysaccharide (Akhter et al. 2015). Prebiotic
act as immunostimulants as they can interact with pattern recognition receptors (PRRs) present
on macrophages (Bron et al. 2012).
Synbiotics are a combination of prebiotics and probiotics which can be provided as dietary
components to promote the health of individual organisms, help in the colonization and
maintenance of dietary microbial supplements in the gut, and promote the growth of beneficial
microbiota to improve the host health (Cerezuela et al. 2011, Gibson and Roberfroid 1995).
The following session discusses how probiotics, prebiotics, and synbiotics can benefit shrimp
aquaculture.
Probiotics, prebiotics, and synbiotics to combat infectious agents
Probiotic bacteria can activate the immune system of shrimp to prevent infections. A probiotic
bacteria M146 administered orally reduced shrimp mortality and showed an
immunostimulatory effect in shrimp by downregulating the genes of IMD and Toll signaling
pathways. On the contrary, W1B, another probiont did not display any immunostimulatory
effect but acted as an effective antimicrobial and quorum quenching agent against V. harveyi
(Yu et al. 2020). Khademzade et al. (2020) found that the addition of probiotic bacteria
Bacillus cereus in rearing water showed better immunostimulatory effects in shrimp reared in
the earthen pond with an enhanced lysozyme activity and increased total haemocyte count.
Hence, probiotics act similar to vaccines by triggering the immune system, due to which they
can be utilized as prophylactic agents.
Probiotic bacteria can compete for space and nutrition, thus excluding these pathogens from
the shrimp intestine (Dash et al., 2017). Feeding shrimp with B. cereus caused extensive
colonization of the probiont in shrimp and significantly reduced the population of
V. alginolyticus and V. parahaemolyticus in the intestine (Vidal et al. 2018). The shrimp fed
with a freeze-dried probiotic consortium including Bacillus subtilis BUU 005, Bacillus
polymyxa BUU 003, Bacillus megaterium BUU 002, B. licheniformis BUU004,andBacillus
thuringiensis BUU001 showed a significant reduction in the colonization of
V. parahaemolyticus and V. cholerae in the gut of juvenile shrimp (Nimrat et al. 2019).
B. subtilis strain IPA-S.51 and Shewanella algae strain IPA-S.252 provided in shrimp feed
reduced the load of Vibrio sp. in the hepatopancreas of shrimp (Interaminense et al. 2018).
Dietary administration of Lactobacillus sakei strain FB011 displayed immunomodulatory
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effect and anti-Vibrio effect in shrimp (Le and Yang 2018). Probiotic supplementation of
Bacillus PC465 in feed, boosted the resistance of shrimp to WSSV infection (Chai et al. 2016).
The intestine of healthy shrimp is a natural reservoir of symbiotic bacteria that can be used
for isolating probionts. Pseudoalteromonas sp. CDM8 and CDA22 isolated from the hindgut
of healthy shrimp were potential probiotic candidates for controlling AHPND (Wang et al.
2018). B. subtilis AHAHBS001 isolated from healthy shrimp of coastal areas displayed
significant resistance to AHPND strains of V. parahaemolyticus upon addition of the probionts
in the feed (Kewcharoen and Srisapoome 2019).
Probiotics can modulate the gut microbiota for providing resistance to microbial infections
in shrimp. Probiotic bacteria Streptomyces sp. RL8 enriched feed had a protective effect on
P. vannamei upon challenge with V. parahaemolyticus. Statistical Analysis of Metagenomic
Profiling (STAMP) revealed that the probiont stimulated antimicrobial producing
Bacteriovorax population of shrimp gut, which provided resistance to infection (Mazón-
Suástegui et al. 2019).
Prebiotic supplements can alter the gut microbiome composition by favouring the growth of
beneficial microbes (Gibson and Roberfroid 1995). Li et al. (2007) administered short-chain
fructooligosaccharides in shrimp feed for 6 weeks in shrimp and found that the prebiotic
supplementation significantly improved total haemocyte count, haemocyte respiratory burst
and altered the microbiome of the gastrointestinal tract. Mannooligosaccharides obtained from
copra meal showed immunostimulatory effects in shrimp by increasing the expression of anti-
lipopolysaccharide factor, penaedin, crustin, peritrophin, and lysozyme, which improved the
survival rate of shrimp upon exposure with V. harveyi (Rungrassamee et al. 2014). Dietary
administration of inulin and mannan oligosaccharide together in shrimp also has been proven
to upregulate immune-related genes like STAT, antilipopolysaccharide factor, crustin,
prophenoloxidase. P. vannamei challenged with V. alginolyticus and WSSV showed enhanced
resistance under this dietary scheme concluding their strong immunomodulatory action (Li
et al. 2018). Bioencapsulation prebiotics using Artemia can enhance colonization of probionts
in the shrimp, and this can be achieved by incubating the nauplii in a mixed solution consisting
of prebiotic and docosahexaenoic acid (Hoseinifar et al. 2015).
Synbiotics have been extensively studied in the field of human and veterinary medicine as
an alternative to fight infectious diseases (Cheng et al. 2014). Several studies have been
conducted to analyze the effects of synbiotics in shrimp aquaculture in controlling infectious
diseases. Huynh et al. (2018a) found that shrimp fed with a synbiotic diet consisting of
Lactobacillus plantarum and galactooligosaccharide showed enhanced immune resistance to
V. alginolyticus infection associated with increased levels of valine, inosine monophosphate,
and betaine in hepatopancreas. Later, it was proven that this diet resulted in a reduced
population of V. harveyi and Photobacterium damselae and enhanced the colonization of
L. plantarum in the intestine (Huynh et al. 2018b). Oral administration of a synbiotic
comprising Bacillus sp. D2.2 and sweet potato extract was found to lower the infection level
of V. harveyi in shrimp (Harpeni et al. 2017). Oktaviana et al. (2014) formulated a synbiotic
diet containing prebiotic oligosaccharides extracted from sweet potato and probiont
V. alginolitycus SKT-bRcapable of preventing co-infection of Infectious Myonecrosis Virus
(IMNV) and V. harveyi in shrimp.
Synbiotic preparation in dry forms are preferable than fresh preparations as they provide
longer viability of the probiont, and this can be accomplished by encapsulation of synbiotics
(Ross et al. 2005). Artemia encapsulated synbiotics containing Pseudoalteromonas piscicida
1Ub, and mannan-oligosaccharide exhibited superior immune response and resistance to
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V. harveyi in shrimp, compared to the diet consisting bio encapsulated probiotic or prebiotic
(Hamsah et al. 2019). A micro-encapsulated synbiotic diet consisting of Bacillus sp. NP RfR
and oligosaccharide of sweet potato was found to render resistance to V. harveyi infection
(Munaeni et al. 2014). Similarly, a micro-encapsulated combination ofBacillus sp. NP RfRand
mannan oligosaccharide augmented immune response to WSSV infection (Febrianti and
Yuhana 2016). These results imply that synbiotics based diet can be employed to control
infectious in shrimp farms and hatcheries (Table 4).
Prolonged use of probiotics has been linked to cause immunosuppression (Sakai 1999), but
integrative use of probiotics with pond bottom soil improvement has proven to reduce the
incidence of Vibrio sp. in rearing systems, increase the survival of shrimp and maintain water
quality in an extensive P. monodon culture system (Pantjara and Kristanto 2020). Probionts
isolated from the terrestrial ecosystem may not give expected results in aquatic animals;
therefore, probiotic strains can be isolated from a marine ecosystem. The challenge in
exploring marine probionts is the difficulty to culture these organisms under in vitro conditions
(Ninawe and Selvin 2009). The studies reviewed here have disclosed the ability of probiotic
bacteria to control only bacterial and viral infections in shrimp. The effect of probiotic bacteria
Lactobacillus plantarum FNCC 226 in controlling fungal infection of S. parasitica in catfish
was reported by Nurhajati et al. (2012) and the effect of this probiotic bacteria can be assessed
in shrimp larvae since they are also susceptible to this fungal infection.
Role of probiotics and synbiotics in water quality improvement
The environment in which shrimp reside is also favourable to pathogenic microorganisms,
hence the chances of acquiring diseases are high and improved immunity is necessary for
survival (Decamp et al. 2008). Probiotic bacteria can be used to eliminate potential pathogens
from rearing systems and reduce the incidence of infection in shrimp farms. Shrimp fed with
probiotic strains B. licheniformis strain LS-1 and Bacillus flexus strain LD-1 showed stimu-
lated immunity and resistance to infections. In addition to this, the population of Vibrio sp. and
the amount of nitrogenous compounds were significantly lower in the rearing water systems
upon probiotic supplementation (Cai et al. 2019). Although the intestinal microbiota acts as a
major source of probiotics, beneficial bacterial strains with probiotic potential can be isolated
from other ecosystems as well. Probiotic strains L. plantarum (JK-8) and Lactobacillus
hilgardii (JK-11), isolated from shrimp ponds, improved the quality of water in shrimp rearing
ponds, and their cell-free supernatants exhibited significant antimicrobial properties against
shrimp pathogens V. parahemolyticus, V. harveyi,andE. tarda (Ma et al. 2009).These
bacteria were also capable of removing toxic wastes such as ammonia, nitrite, and nitrate
from the shrimp rearing system, highlighting their possible use in water treatment for aqua-
culture. Purple non-sulfur bacteria Rhodobacter sphaeroides strains SS15, S3W10, and
Afifella marina STW181 were also capable of reducing the levels of ammonia, nitrite, and
nitrate in rearing and increased survival of shrimp challenged with AHPND strains of
V. parahaemolyticus (Chumpol et al. 2017)(Table5).
A synbiotic diet consisting of B. subtilis and fermented rice bran reduced total Vibrio count
in the rearing water, besides improving the immune response and growth performance of
shrimp juveniles (Moustafa et al. 2020). Hence, probiotics and synbiotics can be utilized to
remove toxic nitrogenous compounds from rearing systems. Yet, the addition of probiotic
bacteria in rearing water is met with several challenges. For example, the possibility of
probiotic organisms altering the microbial consortia of natural ecosystems needs to be
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Table 5 Probiotics, prebiotics, and synbiotics for antimicrobial therapy in shrimp aquaculture
Probiotics Constituent Pathogen Dosage Period of
administration
References
M146 probiotic bacteria V. harveyi 1.0 × 107CFU/gofshrimp 15days Yuetal.(2020)
B. cereus -10
6CFU/mL Added once in
rearing system
Khademzade et al.
(2020)
B. cereus V. alginolyticus and
V. parahaemolyticus
1.0×108CFU/g of shrimp 14 days Vidal et al. (2018)
B. subtilis BUU 005, B. polymyxa BUU 003, B. megaterium BUU 002,
B. licheniformis BUU 004, and B. thuringiensis BUU001
V. parahaemolyticus
and V. cholerae
3.87 ± 0.60–6.80 ± 0.75 ×
108CFU/g of shrimp
90 days Nimrat et al. (2019)
Pseudoalteromonas CDM8 and CDA22 V. parahaemolyticus
(AHPND)
Both probionts fed at 107
CFU/kg of shrimp
21 days Wang et al. (2018)
B. subtilis AHAHBS001 V. parahaemolyticus
(AHPND)
1×10
5–1×10
9CFU/kg of
shrimp
5 weeks Kewcharoen and
Srisapoome (2019)
B. subtilis strain IPA-S.51 and S. algae strain IPA-S.252 Vibrio sp. 106CFU / g of shrimp 35 days Interaminense et al.
(2018)
Bacillus PC465 WSSV 107cells/g of shrimp 30 days Chai et al. (2016)
Prebiotics Short-Chain Fructooligosaccharides - 0.8% 6 weeks Hoseinifar et al.
(2015)
Copra meal derived Mannooligosaccharides V. harveyi 3 g/kg 6 weeks Rungrassamee et al.
(2014)
Inulin and mannan oligosaccharide V. alginolyticus and
WSSV
5mg/g 28days Lietal.(2018)
Synbiotics L. plantarum 7-40 and galactooligosaccharide V. alginolyticus 0.4% of prebiotic and 108
CFU/ kg of probiotic
60 days Huynh et al.
(2018a)
V. alginolitycus SKT-bRIMNV and V. harveyi 1% probiotic and 2%
prebiotic of feed weight
30 days Oktaviana et al.
(2014)
Bio-encapsulated P. piscicida 1Ub, and mannan-oligosaccharide V. harveyi 106CFU/ mL of probiont
and 12 mg/L of prebiotic
13 days Hamsah et al.
(2019)
Micro-encapsulated Bacillus sp. NP RfRand oligosaccharide of sweet
potato
V. harveyi Probiotic of 108CFU/ mL
and 2% prebiotic
40 days Munaeni et al.
(2014)
Micro-encapsulated Bacillus sp. NP RfRand mannan oligosaccharide WSSV Probiotic and prebiotic in 1:1
(v/v) ratio
30 days Febrianti et al.
(2016)
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investigated. The studies discussed above have reported the ability of probiotic strains in
improving water quality, but the mechanism of action behind this is not yet clear.
Future prospects and conclusion
It is quite clear that we need to address the problems related to the use of antibiotics in
aquaculture, with utmost seriousness. Strict measures have to be taken against the use of
antibiotics in an unscientific way. Most importantly, efforts should be made to educate shrimp
farmers regarding the havocs due to the misuse of chemotherapeutic agents in aquaculture. To
avoid further complications in the future, the use of antibiotics should be minimized as much
as possible.
As many of the commercially available antibiotics are under legislative restrictions in
several countries for aquaculture practices, field application studies to investigate the syner-
gistic combination of phages, nanomaterials, and phytobiotics with antibiotics is difficult
(Culot et al. 2019; Lulijwa et al. 2020). But some of these alternatives can be coupled and
analyzed in aquaculture species for synergistic activities. The use of nanoparticles has im-
proved the rapidness of diagnostic methods, but suitable measures to safely discard them need
to be dealt with in the future. The implications in the microbiome of shrimp pertaining to the
use of nanoparticles for treatment need to be studied apart from its antimicrobial potential to
ensure their reliability as therapeutical agents. Phage display technology has been explored
only against WSSV amongst aquaculture viral pathogens, while there are other emerging
pathogens like IMNV, taura syndrome virus and yellowhead virus where this technology can
merit the diagnosis and therapy.
The immune response of oral vaccines was found to be dissimilar between common carp
and Indian major carps (Azad et al. 1999); whether such disparities can exist with the use of
biofilm-based vaccines in various shrimp varieties demands further research. There is a vast
availability of published data regardingthe applicationof probiotics,synbiotics, nanomaterials,
and algal extracts in the prevention of Vibrio and WSSV infections, while there are no reports
available regarding the application of these in the prevention of other viral, fungal, and
protozoal diseases in shrimp.
Phytobiotics and algal extracts are comparatively safer than antibiotics to be supplemented
in shrimp feed as they are of natural origin. Microorganisms might develop resistance to these
extracts if only one active ingredient is used, which is why natural extracts with multiple active
components are preferred (Gupta and Birdi 2017). There is very limited information in the
literature discussing the possibilities of antimicrobial resistance and pollution of the rearing
system due to the continuous use of natural extracts.
The application of probiotics in aquaculture is a safe and effective solution to control
infectious diseases in shrimp. But the performance of probiotics in disease prevention can be
influenced by many factors like stocking density of shrimp in the pond, the prebiotic factors
required for colonization of probionts, and most importantly, the dosage and frequency of
administration (Ninawe and Selvin 2009). Alongside probiotics have to be stringently evalu-
ated for the presence of antibiotic-resistant genes that could get transmitted to other bacteria
before their commercialization (Moubareck et al. 2005). The application of synbiotics is found
to be superior in improving disease resistance in shrimps rather than probiotics or prebiotics
alone. Different combinations of prebiotics and probiotics can be assessed to identify effective
synbiotics. Moreover, bioencapsulation techniques are adopted to improve the efficacy of
1610 Aquaculture International (2021) 29:1591–1620

synbiotics, but the cost of developing encapsulated synbiotic regimens in large scale needs to
be estimated. The effect of synbiotic diets have been extensively studied in P. vannamei and
whether these diets are useful for other shrimp species lacks clarity. Most of the studies
discussed so far have found novel probiotic strains, phytobiotics, or algal extracts and assayed
their antimicrobial potential against pathogens but molecular mechanisms behind the antimi-
crobial activity are poorly studied, which limits further advancement of these innovations into
commercial markets. Forthcoming studies should try on developing antimicrobial components
targeting a wide range of pathogens. It will also be interesting to find out if the bacterial
pathogens develop resistance to any of these antimicrobial components by performing serial
passage assays.
To conclude, it is quite obvious that antibiotics are no longer a reliable chemotherapeutic
agent in the long-run perspective. There is an increased demand in research to find substitutes
for antibiotics, to fight infectious diseases in shrimp aquaculture. So, far research has come up
with some safe and reliable methods which have remarkable outcomes. Although these
methods have been scientifically proven to be effective, they have not been used extensively
in aquaculture farms. The reason behind it can be accredited to lack of studies conducted on
large scale shrimp farms to examine their efficacy, cost, and after effects. It is needless to say
that these strategies are very effective under laboratory conditions, but that does not guarantee
their efficacy in field environments. These approaches should be subjected to thorough
examination in large scale shrimp farms before commercialization. Considering the pace with
which research has progressed till now, it can be expected that these alternatives play a crucial
role in developing sustainable aquaculture practices in upcoming years.
Acknowledgements The authors are thankful to the Department of Biotechnology, Ministry of Science and
Technology, India, for the research grant.
Author contribution PSS and RR performed the data collection, compilation, and preparation of the first draft;
GSK conceived the concept and guided the literature survey synthesis; JS guided the drafting and correction of
the drafts.
Data availability Not applicable
Declarations
Ethics approval and consent to participate Not applicable
Consent for publication Not applicable
Competing interests The authors declare no competing interests.
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