Antimicrobial peptides: Possible
therapeutics in aquaculture
Amit Kumar Sinha1, Parisa Norouzitallab2 and Kartik Baruah1
Disease outbreak has been the ma-
jor bottleneck in the successful farm-
ing of fish and shellfish. In recent
years, much attention has been given
to health management using various
forms of immunoprophylactic tech-
niques, such as vaccination, antibiot-
ics and immunostimulants. However,
these practices are now questioned be-
cause of issues, such as tissue residue,
bacterial drug resistance and environ-
mental imbalances created by regular
use of antibiotics.
Vaccines, although commercially
available, generally cannot be used as
a universal disease control measure in
aquaculture. Conventional techniques,
such as selection and hybridization
have been used to produce a number
of resistant fish for broodstocks using
existing resistant individuals in het-
erogeneous fish stocks. However, such
techniques are restricted to within spe-
cies or closely related species and do
not allow for the employment of po-
tentially effective resistance genes pos-
sessed by other organisms. In contrast,
modern approaches – recombinant
DNA technology, for instance – en-
able the transfer of genes from diverse
sources, such as insects, to improve the
natural resistance of fish to infections
in a directed fashion. However, their
use at farm level is yet to be answered.
This indicated that the above-cited ap-
proaches were not able to solve every
problem alone. Hence, this called for
the development of new approaches
and technologies suited to improve
health of farmed species and, at the
same time, not be harmful to the en-
vironment. In this article, the potential
of using natural antimicrobial agents
or immunoenhancers in eliminating
diseases in aquaculture is discussed.
It has long been known that the
50 December 2009
innate (non-specific) defense system
includes acute phase proteins such as
C-reactive protein and its homologues.
However, an important facet of this
defense mechanism was revealed two
decades ago by the discovery of ce-
cropins, inducible antimicrobial pep-
tides in the giant silk moth Hyalophora
cecropia. These biochemical defense
peptides possess antimicrobial activity
and, therefore, are considered as natu-
ral antibiotics. Fish also possess anti-
microbial peptides as part of their de-
fense system. They are mainly located
in the mucus layer and help eliminate
pathogens before they pass the skin
barrier. The so-called natural antibiot-
ics seem to be effective as a therapeutic
agent and an antimicrobial in enhanc-
ing innate or non-specific immunity in
fish.
What are Antimicrobial Peptides
(AMPs)?
AMPs are low molecular weight
peptides (size <10 kDa; length 12-
50 amino acids) having a net positive
charge because of the presence of ex-
cess basic lysine and arginine residues
over acidic residues and are generally
defined as broad spectrum cationic
molecules. They fold, owing to the
presence of disulphide bridges or con-
tact with membranes, into three-di-
mensional amphiphilic structures. This
leads to the separation of the positive-
ly charged and hydrophilic domain(s)
from the hydrophobic domain(s). Such
a molecule is well suited for interact-
ing with membranes, especially bacte-
rial membranes with their negatively
charged and hydrophilic head groups
and hydrophobic cores. However, some
anionic forms have also been reported
(Vizioli and Salzet 2002).
Occurrence of AMPs
Antimicrobial peptides occur in
both plants and animals, especially in-
vertebrates that lack T lymphocytes or
specific immunoglobulin. More than
750 different AMPs have been identi-
fied in plants and animals, including
humans. Major sources are insects,
chelicerates, crustaceans, ascidians,
molluscs, amphibians and mammals.
In vertebrates, they are usually associ-
ated with peripheral blood leucocytes
or mucosal surfaces. Besides these,
bacteria themselves produce AMPs
and about 50 of them have been iso-
lated from various Gram-positive bac-
teria, especially lactic acid-producing
organisms. Most of these peptides are
synthesized as a prepropeptide. It con-
sists of an N-terminal signal sequence
that aids in targeting of endoplasmic
reticulum, a prosegment and a C-ter-
minal cationic peptide that demon-
strates antimicrobial activity after it
is cleaved from the rest of the protein.
The major advantages of AMPs over
other therapeutic agents are:
1. Broad spectrum of activity against
micro-organisms,
2. Low toxicity for eucaryotic cells,
3. Ease of synthesis, inasmuch as it
does not require specialized cells
or tissues and
4. Rapid diffusion rates, enabling
them to be mobilized quickly to
wounded or infected sites.
Mode of Action
The exact mechanism of action of
AMPs is still a matter of controversy,
although it was originally proposed
that permeabilization of the bacterial
cell membrane was the sole mode of
action. However, evidence has indicat-
ed that some AMPs exert their effects
through alternative modes of action or
that they may, in fact, act upon mul-
tiple bacterial cell targets. Regardless
of their precise mode of action, the ac-
tivities of AMPs are almost universally
dependent on interaction with the bac-
terial cell membrane.
Membrane-Permeabilizing
Mechanism
The first step in this interaction is the
initial attraction between the peptide
and the target cell. Electrostatic bond-
ing between the cationic peptide and
negatively charged components present
on the outer bacterial envelope is the
main driving force for interaction. The
negatively charged components are the
phosphate groups within the lipopoly-
saccharides of Gram-negative bacteria
or lipoteichoic acids present on the sur-
faces of gram-positive bacteria.
In case of Gram-negative bacteria,
peptides are inserted into the outer
membrane structure mediated by hy-
drophobic interactions and involve
prefolding of the peptides into a mem-
brane-associated structure. This alters
the outer membrane structure and per-
meabilizes this membrane to other pep-
tide molecules in a process termed self-
promoted uptake. This results in the
influx of peptides at the cytoplasmic
membrane where they enter the inter-
facial region of the membrane, between
the hydrophilic and hydrophobic por-
tions of the membrane. This step is car-
ried out by electrostatic and hydropho-
bic interactions (Figure 1). The higher
proportion of negatively charged lipids
on the surface monolayer of the bacte-
rial cytoplasmic membrane plays an im-
portant role in the selectivity of AMPs
for bacterial cells over eukaryotic cells,
in which uncharged lipids predominate
at the host cell surface.
Non-Membrane-Permeabilizing
Mechanism
There are several AMPs that show
their action without permeabilizing the
membrane. They translocate across the
membrane and accumulate intracellu-
larly, where they interfere with several
essential cellular processes and mediate
cell destruction. Their modes of action
include inhibition of nucleic acid syn-
Fig. 1. Pictorial description of the self-promoted uptake of cationic peptides across
the outer membrane. The peptide monomers bind to the cell membrane in a α-helical
confirmation (A), this is followed by the localization of more peptide molecules on the
cell membrane (B) after which the peptide helices insert themselves into the hydro-
phobic core of the membrane (C). Progressive recruitment of additional monomers
increase the pore size causing leakage of cytoplasmic material (D) and, hence, death
of the cell (Hancock 2001 and Reddy et al. 2004).
thesis, protein synthesis, enzymatic ac- 1. α Helical AMPs: Linear, mostly
tivity and cell wall synthesis. Buforin II, helical peptides without cystine
an AMP present in frogs, translocates residue, with or without a hinge
across the bacterial membrane without region. Cecropins,magainins and
causing permeabilization and binds to bombinins are in this group.
both DNA and RNA within the cyto- 2. Cysteine rich AMPs: They are rich
plasm of E. coli (Park et al. 1998). Simi- in cysteine residues that are joined
larly, pleurocidin, a fish-derived AMP by with two or more disulfide
and dermaseptin isolated from frog bridges. Defensins are well known
skin, causes inhibition of DNA and representatives of this group.
RNA synthesis without destabilizing 3. β-Sheet AMPs: They generally
the membrane of E. coli cells (Patrzykat form a single β-hairpin structure
et al. 2002). Inhibition of nucleic acid and are approximately 20 residues
synthesis has also been demonstrated long, containing one or two disul-
for AMPs, such as human defensin, fide linkages. They include horse-
HNP-1 and the extended-structure bo- shoe crab peptides, tachyplesins
vine peptide indolicidin (Subbalakshmi and polyphemusin II.
and Sitaram 1998). Additionally, some 4. AMPs rich in regular amino acids:
of these peptides have been shown to These include linear peptides with-
interfere with protein synthesis. Pleuro- out cysteine residue but with a high
cidin and dermaseptin can block tritiat- number of regular amino acids. For
ed leucine uptake in E. coli, and PR-39- instance, attacins and diptericins
and indolicidin-treated cells also exhibit from this group contain glycine-
reduced rates of protein synthesis (Sub- rich and proline-rich domains, while
balakshmi and Sitaram 1998) indolicidins and tritripticin are rich
in tryptophan. Bactenecins Bac-5
Classification of AMP and Bac-7 isolated from bacteria
AMPs are broadly classified into are rich in proline residue.
five groups 5. AMPs with rare modified amino ac-
World Aquaculture 51
 ids: These are the molecules derived
from larger peptides by posttrans-
lational modification or alternative
splicing. Nisin is one such peptide
produced by bacteria Lactococcus
lactis and is composed of rare
amino acids, including lanthionine,
3-methyllanthionine, dehydroala-
nine and dehydrobutyrine.
AMPs Isolated from Fish
In fish, AMPs are mainly confined
to the mucus layers. The early discov-
ered AMPs from fish, such as the red
sea moses sole, Pardachirus marmoratus,
was pardaxin. It is a 33-amino-acid pore
forming peptide with a helix-hinge-helix
structure similar to cecropin and mel-
litin. It is an excitatory toxin that pos-
sesses high antibacterial activity and
low hemolytic activity towards human
red blood cells compared with mellitin.
Lemaitre et al. (1996) isolated two novel
antibacterial proteins, 31 kDa and 27
kDa, from the skin mucosa of the com-
mon carp, Cyprinus carpio, and found
strong bactericidal activity against fish
pathogens. Cole et al. (1997) reported
pleurocidin, a novel 25-amino acid
residue linear AMP, found in the skin
mucous of the winter flounder, Pleu-
ronectes americanus. Park et al. (1997)
isolated misgurin from mudfish, Misgur-
nus anguillicaudatus. It is 21-amino-acid
compound having strong antimicrobial
activity against various microorganisms
and slight hemolytic activity. Addition-
ally, three antimicrobial proteins were
isolated from catfish skin in 1998. The
molecular masses of the proteins were
15.5, 15.5, and 30 kDa. These proteins,
which are closely related to histone
H2B proteins, can inhibit the growth
of Aeromonas hydrophila and Sapro-
legnia spp. An antimicrobial compound,
squalamine, was isolated by Zasloff’s
group from the stomach as well as from
various other organs of the dog shark,
Squalus acanthias. Although a handful
of antimicrobial steroids have been iso-
lated from plants, squalamine is the first
antimicrobial steroid isolated from an
animal.
AMPs Isolated from Shellfish
Recently, it was demonstrated that
the AMPs are the widespread mecha-
nism of host defense in shrimp, mussels
52 December 2009
and crabs. Penaeidins is an AMP docu-
mented in shrimp, such as hemocytes
of Penaeus vannamei. The molecular
weight of penaeidins is 5.5-6.6 kDa.
They have an N-terminal proline-rich
domain and a C-terminal domain con-
taining six cysteine residues. Penaeidins
have several isoforms and are classified
into penaeidin 2, penaeidin 3 and pe-
naeidin 4 according to their similarity
of amino acid sequence. Penaeidin 3
is the most abundant at both levels of
peptide and mRNA in Litopenaeus van-
namei haemocytes. Moreover, different
members of penaeidins family, includ-
ing penaeidin-1, -2, -3a, -3b and -3c
have been described. These molecules
have chitin-binding properties as well as
antimicrobial activities against Gram-
positive bacteria and filamentous fungi.
They also have the property to opsonise
bacteria of the genus Vibrio.
Another AMP, crustin, was first iso-
lated from the shore crab Carcinus mae-
nas. It is cysteine-rich 11.5-kDa pep-
tide with antimicrobial activity against
Gram-positive bacteria. However, the
presence of homologues of crustin in
L. vannamei and the Atlantic white
shrimp L. setiferus was also document-
ed. Vargas-Albores et al. (2004) further
divided penaeid crustins into crustins
I and P. These AMPs are dominantly
synthesized and stored in haemocytes
and their release from haemocytes is in-
duced by bacterial infection. Also, nu-
merous AMPs have been characterized
from mussels recently. They are orga-
nized into three groups. The first group
comprises the defensins. Charlet et al.
(1996) reported two defensins, contain-
ing six cysteines residues from Mytilus
edulis. The second group of molecules,
the mytilins, consists of five isoforms
(A, B, C, D and G1). The isoforms A
and B were isolated from M. edulis and
isoforms B, C, D and G1 from M. gal-
loprovincialis. The third group of pep-
tides contains myticins A and B, which
were characterized from hemocytes and
plasma of M. galloprovincialis.
Limitations
Certain technical difficulties exist
when using AMPs as therapeutics in
aquaculture. The initial binding of the
positive charged peptide to the nega-
tively charged microbial membrane is
a simple electrostatic interaction and
therefore very sensitive to the ionic
strength of the medium. The sensitivity
to ionic strength conditions has serious
implications for proper evaluation of
the antimicrobial potency of peptides.
Peptides from marine animals that are
adapted to living in a salt-rich envi-
ronment are generally less sensitive to
ionic strength. Such peptides have been
found in marine organisms such as
molluscs, tunicates, shrimp, crabs and
various finfish. Little is known of the
structural features that are responsible
for the insensitivity to salt. Physiologi-
cal implication of salt sensitivity could
affect the in vivo activity of the peptide.
One should be very careful to extrapo-
late in vitro phenomena to physiological
processes and always bear in mind that
the situation in vivo is much more com-
plex than a simple test in vitro.
Conclusion and Future Role in
Aquaculture
This discussion indicates that
AMPs, which have a startling range of
antimicrobial activities against most
Gram-negative and Gram-positive
bacteria, fungi, enveloped viruses
and eukaryotic parasites as well as
an immuno-modulation effect, might
be useful as promising antimicrobial
agents in aquaculture. Hitherto, there
have been few examples of the appli-
cation of AMPs as therapeutic agents
in aquaculture though many are an-
ticipated. Jia et al. (2000) observed
cationic AMPs protect coho salmon
(Oncorhynchus kisutch) against infec-
tion caused by Vibrio anguillarum.
Japanese medaka injected with ce-
cropin B homolog transgene AMP
showed more resistance to pathogenic
bacteria such as P .fluorescens and V.
anguillarum compared to control fish
(Sarmasik 2000). Hence, all of these
peptides seem to be extremely effective
therapeutic agents in aquaculture but
elucidation of their biological impor-
tance in innate immunity and realiza-
tion of their full clinical potential will
require much more effort.
Before these new agents can be ap-
plied in aquaculture, there are some
important topics that should be ex-
plored. First, the impact of AMPs on
the virulence of several aquatic patho-
gens should be studied in detail to de-
termine if they are valid antimicrobial
agents for aquaculture. Secondly, the
effect of such agents on the health of
the final consumer of the aquaculture
product and also on the aquaculture
system should be determined. More-
over, some practical problems, such as
the cost of treatments and the delivery
system of such agents to the site of ac-
tion, should be considered. Finally, the
problem of eventual resistance devel-
opment should not be neglected.
Notes
1
Laboratory of Aquaculture and Artemia
Reference Center, Department of Ani-
mal Production, Ghent University,
Gent, Belgium
2
Department of Fisheries and Environment,
Faculty of Natural Resource Engineer-
ing, Tehran University, Karaj, Iran
*
Corresponding author: baruahkartik4@
rediffmail.com
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World Aquaculture 53