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ANTI MICROBIAL DEFENSES IN MUCUS OF ATLANTIC SALMON, OLIVE
FLOUNDER ATLANTIC COD AND CHANNEL CATFISH.
NAME : FARIHA KIBRIA
STUDENT NUMBER : 999857141
SUPERVISOR: DR.TEREBIZNIK

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ABSTRACT:
Fish mucus is the outermost layer and first line of defense. Outermost layer of fish skin does not
contain keratin as it leaves in an environment filled with pathogens. Fish mucus contains
enzymes like lysozymes, alkaline phosphotase , trypsin and amp as defense mechanism. Amp in
fish serves various functions which were studied in this paper
Introduction
Skin or the integument system is the largest organ of most living organisms. It is a multi-
function organ that covers the body and protects it from the entry of pathogens and prevents
leakage of water, nutrients or solutes. Apart from playing a crucial role as a barrier and first line
of defense, it also serves multiple other functions such as physical and chemical protections,
osmoregulation, sensory activity, hormone metabolism.( Rakers et al, 2009). Even though skin
is present in all living organism, the anatomy of the skin varies among different animal.
Mammalian skin is the largest organ of body. The texture of the mammalian skin is usually
dry and soft in uninfected state. It is composed of two different layers epidermis and dermis
which serves various functions. The epidermis layer is composed of non-vascular dead cells
known as Stratum corneum(Sc). It provides physical, biochemical and adaptive immune
protection. In undamaged state, this layer prevents water loss, foreign substances from entering
the body and also helps to protect the underlying organs , muscles and nerve endings from
injury. Epidermis also consist of Langerhans cells, macrophage and antigen presenting cells
which are involved in immune system(Prokosh et al, 2008). This cells together form Skin
Associated Lymphoid Tissue(SALT) where antigen presentation and immune response initiation

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occurs. Sc forms a continuous layer of protein rich cells called keratinocytes from which
keratin arises. Interaction of keratin with a matrix protein called flaggerin consists of 80-90% of
epidermal mass (Prokosh et al, 2008). The epidermis layer composed of mainly involved in
protection and the dermis layer is richly supplied with blood vessels (Martin-Granodos and
Mccaig ,2014). . Beneath the epidermal layer lies tight junctions which regulate the movement
of ions and solutes( Kubo, 2014). In mammalian skin , mucus membrane in epidermis layer is
absent unlike in fish skin ( Figure 1). Fish mucus is ideal for protecting the animal from various
pathogens present in the water. Mucus membrane is seen in mammals in areas which are
constantly exposed to pathogens like gut, oral cavity, nose, urogenital tract and respiratory tracts.
This plays a crucial role in preventing pathogen attachment in these organs .
The layer of tissues just below the epidermis is known as dermis which is rich with blood vessel
and also contains nerve endings for different senses, hair follicles , sweat and mucus gland . The
dermis is a thick layer of flexible tissue that provides the strength and flexibility. The innermost
layer of is known as hypodermis. This layer is composed of fat and connective tissues . Fat
provides the body with insulation against adverse temperatures and padding for the delicate
internal organs. It is also an energy storage region [Prokosh et al, 2008].

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Figure 1: Structure of mammalian skin. It can be noted that in Mammalian skin, the epidermis
is composed of keratinocytes and dead cells. Also mucus glands are not prominent in mammal
skin. This figure has been adapted from McCaig, C. M.-G. (2014).
In fish, skin is organised differently. It can be noted that unlike mammals, fish epidermis is not
composed of dead tissues. Mucus membrane is the outermost layer of the skin in fish and it
contributes to the moistness of the fish skin. This layer has goblet cells which secretes viscous
peptide known as mucin. The viscosity of the mucin helps to trap pathogen that try to invade
through the mucus membrane( Rakers et al, 2013). Mucus associated Lymphoid Tissue can be
subdivided into Gut Associated Lymphoid tissue( GALT), Skin Associated lymphoid
tissue(SALT) ( Cesta M.F., 2006). Even though the mucosal immune system of fish and humans
is comprised of different type of lymphoid tissue ,these tissues share some common mechanisms
to fight against pathogen (Rakers et al, 2013). It has been proven that in presence of stress,

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mucus secretion of the fish rapidly increases. Mucus was also noted to be contain many
antimicrobial products such as alkaline phosphotase, lysozymes, Cathepsin D, transferrin (Hellio
et al., 2002 and Rakers et al,2013). Scales observed in fish skin are part of immune system
which originate from the dermis. The dermis of fish skin also contains gland like human skin.
However they are not sweat or sebaceous glands but glands involved in self defense. Among
different defense mechanisms and peptides present in the skin of mammals and fish are
immunoglobulin, anti-microbial peptide, lysozymes etc. This paper will focus on the defense
mechanisms present in the mucosal layer of fish.
Figure 2: Schematic representation of the fish skin. In fish skin mucus layer ( depicted by
mucus glands ) is the outermost layer of the skin. The skin is composed of three layer Epidermis,
Dermis and Hypodermis. Fish epidermis lacks keratinocytes which are the common constituents
of mammalian skin. This figure has been adapted from Rakers et al, 2013
Fishes activate their defense mechanism when there is stress present in their environment. In
optimal conditions, there is lower level of activation of defense mechanism. Optimal condition
can be defined as the environment that the fish is most comfortable in. Optimal condition varies
among different fish species. For example marine fishes would exhibit symptoms of distress
when placed in fresh water and vice versa. The fish species studied in this paper are Atlantic

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Salmon, Atlantic Cod , Channel Catfish and Olive flounder. Channel Catfish fare well in clear
water where as Salmon struggles in cloudy water ( Ramesh, 2013). The natural habitat for
Atlantic Cod is marine brackish water and fresh water and for Olive Flounder it is marine
water. The mucosal immune system in fish is composed of different defense immune
components with bacteriostatic and bactericidal activity such as protease enzymes, complement
proteins , lysozyme, antimicrobial peptide, antibodies and lectin.
Figure 3: Schematic representation of the action of mucus and AMP in response to injury.
In situations of mechanical or chemical injury, the epithelial cells secrete cytokines like IL-IB to
aide in tissue recovery. SECRETION OF CYTOKINES ATTRACT NEUTROPHILL AND T
CELLS WHICH AID IN WOUND HEALING. Also the goblet cells in the epidermis secretes
mucus which contains AMP, lysozymes, enzymes which interfere with DNA/RNA protein
synthesis of the microbe. This figure was made based on ideas from Rakers et al, 2013
Immunoglobins or antibodies are proteins secreted by the body in the cell surface in response to
bacteria, viruses or other pathogens. There are five of antibody present and serves a different
function. Antibodies are highly specific for the type of pathogens secreted. When an antigen is
Attack by bacteria, virus
or other pathogens
resulting in chemical or
mechanical injury
Secretion of IL-IB in response to
injury which attract neutrophills
Mucus issecreted by goblet cellsin response
to injury. AMP containlysozymes, alkaline
phosphotase, AMP like pleurocidin, defensins
AMP interfereswith DNA/RNA
protein synthesisto prevent
bacterial growth

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present, immune system secretes an antibody specific for it. Binding of the antibody to a
pathogen helps the immune system to destroy the pathogen. In mammals there are five different
isotopes of antibody present and each type has a specific function. However the type of
immunoglobulin in fish is different compared to mammals. According to previous research, it
has been noted that all skin cells across families have same origin (Rakers et al, 2008). The
difference in skin originated is due to adaptations to sustain in their respective environment. In
teleosts, the oldest fish class of vertebrates the skin represent more closely to gut tissues.
According to Sunyer et al, the mucus layer in the fish elicits a gut like mucosa instead of skin
like mucosa. In mammals IgG is the most common type of antibody secreted by the body and
the most common type of antibody in the mucosa is IgA. According to Sunyer et al, teleost skin
expresses IgT an immunoglobins that is specialized in gut like immune response and it reacts
with the pathogens present in the fish skin. IgT/Z has been found to be similar to IgA, the isotype
of antibody found in mammalian skin.
Table1: Isotypes of Antibodies and their function in mammals. This table was created
based on ideas from Rakers et al, 2014.
Name Description
IgT/Z Teleost specialized immunoglobins that are present in the skin mucus of
fish. It is similar to mammalian IgA
IgM The most prevalent form of antibody. It is involved in systemic
immunity.
IgD Found in fish plasma

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However research has shown that the fish skin is similar in structure compared to its gut . Since
mammalian gut is always exposed to stress factors in form of acid , microbes and toxin digested
with food, it can be speculated that mammalian gut like have similar structure to fish skin. It can
be noticed that there is an absence of avascular keratinocytes containing epidermis( refer to
figure 3) in gut surface of mammals. In gut tissues, the outermost layer is mucus membrane and
epithelial cells lie below it. This particular composition helps the gut to fight against toxins,
pathogens that enter the gut through food .
Figure4: Gut Associated lymphoid Tissue. Since gut is constantly exposed to microbes, mucus
membrane is the outermost similar to fish skin. The goblet cells present are involved in secretion
of mucus.
Other than immunoglobins, there are other defense associated peptides present in the fish mucus
such as Anti-microbial peptide, lysozyme, proteases, Trypsin.
Anti-microbial peptides

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Antimicrobial peptides are key components of innate immunity. Peptides present in human
skin are derived form neutrophils, keratinocytes, sweat glands and sebaceous glands. Some of
these proteins are transcribed constitutively whereas other peptides are induced in presence of a
pathogen. These agents are active against a broad range of pathogens. Antimicrobial peptides
mostly in fish are made from biologically active pro-proteins but some AMP are made from
larger functioning proteins. These proteins are able to inhibit DNA, RNA and protein synthesis
of invading pathogen in fish skin. They are also involved in wound healing and cytokine
response. After the discovery of pleurocidin in winter flounder, a wide range of anti-microbial
peptide with variable function has been found. Below only the peptides found in the above
mentioned fish species will be discussed.
Piscidine and Pleurocidin
Piscidine and pleurocidin are a member of a linear, amphipathic family of polypeptide containing
similarity with insect and mammalian AMP. The first member of these class of defense
molecules were discovered in winter flounder. Piscidines are found usually in gills, skins and
intestines of fish species (Su-Fang Niu, 2013) but in Atlantic Cod it is ubiquitous being present
in heart, oocytes, heart, exocrine and endocrine glands, swim bladders etc[ Ruangsri et al, 2012].
It has also been noted in immune cells and granulocytes. The piscine genes are induced by
various bacterial stimuli like bacterial effectors and LPS. This class of anti-microbial peptide
has been found have a broad class of activity being active against both Gram positive and
negative bacterial species and also possess anti-fungal, anti-viral activity. It was also found to be
highly effective against water mould. Phagocytic Granulocytes have exhibited the ability to
destroy extracellular pathogens by secreting piscidine and thus piscidine is effective against

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both intracellular and extracellular bacteria. From several studies, it has been noticed piscidine
disrupts the plasma membrane of the microbes which results in exocytosis of the intracellular
material[REFERENCE] Use of membrane models however suggested that the composition of
the lipid membrane determines the effectiveness of the peptide. HOW? This peptide is able to
maintain its function even in very harsh conditions such as high salt concentration and
temperature.
Pleurocidin is a short basic peptide found in the mucus of winter flounder. It is usually
localized in the mucin granules and intestinal goblet cells of the fish. This is a very basic
molecule and it forms an amphipathic alpha helix kills bacteria by damaging its plasma
membrane. (Cole, 2000)
Even though Piscidine and pleurocidin is a common polypeptide, it was not observed in the fish
species studied in this experiment ( Diamond, 2014). However there are some defense
associated peptides like defensins , hepacidins and histone derived proteins that were noted in
all fish species studied.
𝜷 −Defensins
Cysteine rich-cationic antimicrobial peptides are known as a defensins. This class of anti-
microbial peptide is noted in plants, fungi, invertebrates and vertebrates. The general
conformation of defensins is cysteine-stabilized α-helical and β–sheet folds. In fish only
𝛽 −Defensins have been observed unlike other animals. Defensins have been observed in both
fresh and marine fish species and it was one of the anti-microbial defenses noted in all fish
species studied. These proteins have shown moderate activity against most Gram-positive and
Gram negative bacteria. Exceptions are highly concentrated Gram-positive Planococcus citreus

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and Gram-negative Aeromonas hydrophila . Defensins are not only active bacteria but also
against fish specific virus such as Singapore grouper iridovirus (SGIV), viral nervous necrosis
virus (VNNV), haemorrhagic septicaemia virus (VHSV). β-defensin genes were induced by
bacterial cell wall component such as LPS, β-defensin and peptidoglycans (Jun-Yan Jin, 2010).
In Olive flounder, presence of pathogens induces multiple β-Defensins for protection.For
Atlantic Cod, β-defensins have also been noted to be capable of stimulating antimicrobial
activity in phagocytes. Defensins are expressed constitutively from early stages as a component
of developing innate immune system. However different isoforms of defensins are present in
fish species and therefore no specific pattern of defensins expression can be noted. The highest
basal expression is noted in the skin of fish and in other immune organs of fish such as head-
kidney tissues and spleen (Jun-Yan Jin, 2010)
Histone derived
Analysis of novel anti-microbial peptide in amphibians resulted in discovery of a protein with
identical N-terminal as histone. This protein is ubiquitous in fish, amphibians and plants. In a
large number of fish species, histone derived AMP has been observed. These proteins exhibit a
broad spectrum activity against fish and mammalian pathogen, water moulds and parasites
( Diamond, 2014). Histone derived peptides are expressed in skin, gills and gut. The expression
of histone derived peptides are driven by stress factors . According to Robinette and Noga
level of stress in channel catfish was measured using levels of Histone Like Proteins( HLP). It
can be noted that the level of HLP decreased significantly in presence of stress for a week.

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Chronic stress over a prolonged period of time resulted in further decrease of HLP (Robinette
D.W., 2001)
Hepcidin
Another cysteine rich short peptide with antimicrobial activity observed in fish is Hepacidin.
This class of peptide was first observed in human and later has been noticed in different fish
species. This peptide is secreted by the liver(Rossi, 2005). The general structure of Hepacidin is
a β sheet composed of hairpin with four sulfide bridges( Ross , 2005 and Howard, 2002). Fish
Hepacidin genes have undergone multiple evolutionary changes and thus there are different
version of genes available with variable units of cysteine. Even though Hepcidin was initially
found to be anti-microbial peptide, it is also the major iron regulating peptide. Even though
hepacidins have been indicated as a potent anti-microbial and anti-viral peptide, the major role
of hepacidins is working as an inhibitor for iron transport regulator protein ferroprotein. Being
an inhibitor for Ferroprotein, it helps to reduce the amount of iron present in the blood plasma
which is essential for the growth of bacteria. When iron levels in the plasma is high, hepcidin
secretion also increases( Rossi,2005). Hepcidin can be induced by both gram positive and gram
negative bacteria as well as virus, fungi and tumor cell lines. Presence of cytokines and bacterial
lipopolysaccharide can also initiate the synthesis and release of this protein. Hepacidin are
induced after 3 hour of bacterial attack and decay quickly afterwards. Hepacidins can also alter
viability of tumor cells against human cancer cell lines ( Diamond, 2014).
Enzymes in fish skin:

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Lysozymes are a class of widely studied constitutively expressed bactericidal enzymes.
This bactericidal enzyme of the immune system has been reported in various fish tissues such
as skin, spleen, liver, mucus, lymphoid tissue and contributes significantly to the protection
against pathogens (Palaksha,2007). It acts directly on the peptidoglycan wall of gram positive
ghsalmon, in periods of stress higher level of lysozyme was noted. However in species like olive
flounder, the level was constant. In marine species higher level of lysozymes have been noted
compared to fresh water species as they are constantly in environment with higher amount of
stress(Fast, M.D, 2002)
Alkaline phosphatase is a hydrolase enzymes that removes phosphate from
proteins, nucleoids in alkaline condition((Diamond, 2014). This enzyme was noted in the mucus
of different fish specimen and the level of this enzyme was elevated in periods of stress. In
Olive flounder, the leve of alkaline phosphotase was found to be very low concentration. This
enzyme plays a crucial role in the wound healing and was noted in the mucus of Olive Flounder
and Atlantic Salmon (Palaksha,2007).
Trypsin is a low molecular serine protease (MW 24kD) found to exhibit strong activity
against Gram positive bacteria in the mucus of Atlantic Salmon. Presence of a Trypsin like
molecule was found in the mucus of Olive flounder. This protease could play a strong role in
protecting the Olive Flounder against pathogenic attack( Palaksha,2007). Form of attack can
either be specific working against pathogens or indirectly change the consistency of the mucus
membrane leading to the sloughing of the mucus.
Fish species under focus :

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Olive flounder is a marine cultured fish found in East Asia mainly in Korea and Japan.
Since this fish is mostly cultured, it has a high chance to face toxins and stress such as low
oxygen level, growth of bacteria due to stagnant water. It is of high economic value and therefore
researchers are focusing on its defense mechanisms to fight economic loss . Antimicrobial
peptides such hepacidin and defensins are present in the mucus membrane of this fish.
According to Palakha et al, the mucus also had significant amount of lysozymes, transferrin,
trypsin like proteases. The presence of these defensins are important for fish’s protection in
presence of stress.
Atlantic salmon is a common fish of North America and a popular choice in local diet. It
is also a fish of great economic value. When this fish is in stress, the mucus secretes a wide range
of defense molecules and peptides, notable among these are defensins, hepacidin, pleurocidin
and histone derived proteins (Diamond,2014). In periods of extended stress it has been noted that
stress hormone gets down regulated in this fish. According to Madaro et al, starvation over a
period of three week resulted in reduced cortisol response in presence of stress According to
Firth et al, it was noted that during periods of prolonged stress such as infection with lice results
in increased level of stress hormone like cortisol and enzymes like lysozymes, proteases,
esterase. In cases of very serious infection, the level of alkaline phosphotase also increases. High
amount of salt is a stress factor for many fish species. It has been noted that the level of defense
peptides in salt water is much higher that the level of fresh peptides. Therefore for Atlantic
salmon, environment plays a critical role as a modulator of immune system.
Atlantic Cod is a cold adapted fish available in the ocean. It is a fish of interest
in aquaculture. Genomic analysis of this fish has revealed a unique immune system. Atlantic Cod

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has lost the classical pathway of adaptive immune response. (Star, Nederbragt et al, 2011). Not
only has this fish have an evolved a different pathway for adaptive immune response but they
also has a very high level of IgM in their skin and very low antibody response upon pathogenic
attack [ REFERENCE]. Also a novel defensins has been recognized in this fish. Genes for this
group of anti-microbial peptide was noted in some tissues including skin, swim bladder, sub
mucosa, excretory kidney of fish from early point in development. This protein was found to be
highly effective against gram positive bacteria. (Fernandez et al, 2013). Anti-microbial peptides
are secreted in presence of stress. In the mucus of this fish, major anti-microbial peptides found
along with defensins are hepacidins, histone derived proteins, piscidins (Diamond, 2014). Along
with defense peptides, microbiota plays an important role in fish defense . Following stress such
as captivity or crowding, change in the variety of microbiota of Atlantic Cod has been
noticed(Dhanasiri,2011). Also due to crowding, there was significant increase in bactericidal
enzymes like lysozymes, alkaline phosphotase post stress(Marlowe at al, 2009). Significant
increase in cortisol level following periods of stress was noticed . Also change in the
composition of diet impacted the intestinal microbiota of this fish. When this fish was captivated
from wild environment and fed an artificial diet, intestinal microbiota decreased rapidly
(Dhanasiri, 2011) . Therefore Atlantic Cod has wide variety of defense peptides present in their
mucus and their diet plays a crucial role in their immune response and they have a significantly
different adaptive immune response.
Channel Catfish is a fish of North America. It resides predominantly in lakes of North
America. This fish is important for aquaculture. Mucus of this fish contains AMP like defensins,
hepacidins and histone derived peptides( Diamond, 2014). Secretion of mucus increases in
presence of stress factors. During pathogen attack, feeding in fish breeding facilities is

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sometimes attenuated to prevent spread of the disease. However it has been noted that an
underlying complex relationship between diet and immunity is present. A short period of nutrient
deprived state hampers the innate immunity of this fish . As seen in case of F.columnare
infection, absence of diet significantly altered secretion of critical immune factors in the mucus
in this fish. (Liu et al,2013). . There was significant decrease in innate immunity factors like
lysozyme . Another factor that is important in immunity of fishes specially adaptive immunity
is secretion of antibody.
Various applications of AMP
The aquatic environment contains large amount of pathogens that the fish is exposed to
consistently . Since the skin of the fish is constantly exposed to pathogens, it acts as a first line
of defense. In fish, a moderately developed adaptive immune system is noted due to absence of
very specialized immunoglobins. Fish has a strong innate immune system to make up for lack of
a specialized adaptive immune system( Rajanbabu and Chen , 2011). Fish secrets a wide range
of these Anti-microbial peptide for defense. It has been noted that Anti-microbial peptide is an
important component of the fish innate immune system . Anti-microbial peptides are not only
important component of immune system, they also serve various other functions. AMP
functions against several strains of gram-negative and -positive bacteria. It has been noted that
these positively charged peptides bind to negatively charged molecules present in the membrane
of bacteria. This binding results in pore formation in the bacteria cell membrane and damages
the bacteria. These defense peptides fight against bacterial, viral, fungal infections by either
disruptive aka lytic activity or by pore-forming action( Rajanbabu and Chen, 2011)

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Anti-viral and Anti-fungal
AMP can also function as anti-viral and anti-fungal agents. Β-Defensins have exhibited
antiviral functions against fish specific virus VHSV in rainbow trout. B-defensins have also been
noted to be able to effective against IPNV a fetal virus in salmon . Piscidine-2 has been noted to
act as an anti-fungal against Striped bass fungus. Histone 2B protein in Atlantic Cod has been
noted to be effective against specific fish pathogens(Bergsson G, Agerberth B, Jörnvall H,
Gudmundsson GH, 2005). Also hemoglobin peptides has shown antiparasitic functions against
Ichthyophthirius multifiliis in Channel catfish (Ullal AJ, Noga EJ., 2010).
AMP as immunomodualtor and anti-tumor agent
AMP is also an important immunomodualtor in fish. Absence of AMP has exhibited Crohn’s
disease symptoms. Fish defensins might be able to act as an alternative to defensins deficiency
in mammals( Rajanbabu and Chen,2011). When modulating the effect of AMP on pro-
inflammatory gene , it was noted that it increased the inflammatory response and provided
better protection against pathogenic infection. Also AMP treatment reduced the severity of re-
infection. AMP has also shown ability to reduce growth of tumor cell lines (Chen JY, Lin WJ,
Lin TL, 2009)
AMP as adjuvant in vaccines and in development of vaccines
AMP can be used to induce an immune response along with vaccines. Since , it has been noted
that AMP can induce pro-inflamatory response as seen in study using fish pleurocidin and trout
macrophage cell line, this would serve as a perfect adjuvant with sub-unit vaccines . Also AMP
could be used to silence pathogenic bacteria strains. Since inactivation of bacterial strains by

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formalin results in allergic reaction in certain patients, use of AMP would help to overcome
that( Rajanbabu and Chen, 2011).
AMP in agriculture
It can be used in plants to prevent infections for crops like rice, tobacco, tomato and potato. It
can also be used as a fungicide in plant production. Since fish based AMP is less likely to result
in situations of antibiotic resistance, it can be used as an option to treat infections instead of
conventional drug. Use of AMP transferrin derived transferricin helped to prevent E.coli
infection in milk. AMP can be used to in food processing and packaging in industries. Not only
in agriculture, AMP can also be used in animal husbandry to prevent infection of mammary
gland(Keymanesh,2009)
In fish mucus is the outermost layer in the skin unlike mammals. Since fish is always exposed to
pathogens in water, their skin acts as the first line of defense. Mucus in the skin which is secreted
by goblet cells contains many defense peptides like pleurocidin, hepacidin, histone derived
peptides and defensins. It also contains many bacteriostatic enzymes like alkaline phosphotase,
lysozymes. AMP can also serve as anti-viral, anti fungal, adjuvants, food processing and tumor
cell suppresors and play many important functions in agriculture. Therefore use of AMP in
absence of traditional antibiotics could help to prevent instances of antibiotic resistance due to
irregular intake of the medicine. Due to various potential of this drug , more research needs to be
done to exploit how these defense molecules in fish could help in the immune defense of fish.

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