Download 6 REVIEW OF LITERATURE Crustacean immune system Many

REVIEW OF LITERATURE
Crustacean immune system
Many aspects of the crustacean immune system are now comparatively well
characterized and documented (Smith and Chisholm, 1992; Soerhall and Cerenius, 1998;
Holmblad and Soderhall, 1999; Sritunyalucksana and Soderhall , 2000). Lot of
immunology research has been conducted on crab, crayfish and lobster but subsequent
advance research on shrimps and prawn were still scanty but most of them are
concentrated in their the basic mechanisms (Bachere et al., 1995; Sritunyalucksana et al.,
1999; Sritunyalucksana et al., 2001). The circulating haemocytes play extremely
important roles not only by direct sequestration and killing of infectious agents but also
by synthesis of a battery of bioactive protein molecules (Smith and Chisholm 1992;
Smith and Chisholm 2001). Essentially, the haemocytes execute inflammatory-type
reactions such as phagocytosis, haemocyte clumping, production of reactive oxygen
intermediates and the release of microbicidal proteins There appears to be partitioning of
these between the different cell types, although there may be species differences in the
way this occurs. Importantly, these, antibacterial proteins and opsonins are contained
within or derived from the granular cells, although there may be some contribution made
by the semigranular cells. Certainly, full immune reactivity is always achieved through co
operation and interaction between haemocyte types or their products. A key protein is
peroxinectin and this is present in granulocytes of both Dendrobranchiata (shrimps and
prawns) and Pleocyemata (crayfish and crabs) (Sritunyalucksana et al., 2001). Likewise,
the fundamental role of phenoloxidase seems to be similar in the immune system of
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Penaeus monodon
(Bachere et al., 1995; Sritunyalucksana et al., 1999) and other
decapods. Further, the class of antibacterial peptides known as penaeidins have so far
only been found in species of penaeid shrimp and so would seem to be exclusive to this
group (Destoumieux et al., 2000; Sritunyalucksana et al., 2001;). It is clear that, the
crustacean immune mechanism is generally conserved across the phylum, specific
differences between distantly related species do occur.
Under non-challenge
(i.e.uninfected) conditions, most immuno-reactive factors (e.g. peroxinectin, antibacterial
peptides, clotting components) are stored within the haemocytes, usually in an inactive
state. Their release and ‘activation’ occurs through regulated exocytosis following
stimulation by the presence of non-self molecules in the haemolymph. The ‘clumping’
(i.e. encapsulating) behavior of the haemocytes is influenced by some of the proteins
exocytosed from the cells. Importantly, this has a dual effect; it serves to contain the
spread of any infective particles that may have gained entry to the haemocoel and it also
localizes the response, so that induction of the biochemical pathways that underpin
immunity does not result in massive intravascular inflammation. The biochemical and
molecular signals involved in these events are gradually being elucidated and a number
of key proteins have been purified, sequenced and cloned. Many of these are associated
with the prophenoloxidase (proPO) activating system. This is a cascade of enzymes and
other proteins located in the granular and semigranular haemocytes of decapods that is
activated by the carbohydrate constituents of microbial cell walls, through pattern
recognition binding molecules. Activation and results in the generation of a number of
potent bioactive products which assist phagocytosis, cell to cell adhesion and the
formation of melanin deposits (Soderhall and Smith, 1986; Soerhall and Cerenius, 1998).
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Without doubt, the proPO system is a dominant part of the crustacean defense system
exerting effects on cell behaviour, liberation and/or activation of functionally important
molecules and ‘neutralisation’ of infective agents. Its multiplicity of effects, direct or
indirect, not surprisingly has made it one of the central parts of the crustacean immune
system to be targeted for up-regulation by externally administered ‘stimulants’. This has
probably been fuelled by the early observations of the biochemical character and
functional properties of the proPO system that led to be considered as an ancestral form
of a vertebrate defence pathway, possibly, an invertebrate ‘equivalent’ or forerunner of
the alternate pathway of vertebrate complement (Soderhall, 1982; Ashida and Soderhall,
1984). However, the genes encoding its constituent proteins have been cloned and
sequenced from a variety of species (Soderhall and Cerenius, 1998) and analysis of the
sequence data generated has not revealed any significant homology to the main vertebrate
recognition and immune effector pathways.
Components of Crustaceans Immune system
Invertebrate animals, which lack adaptive immune systems, have developed other
systems of biological host defense, so called innate immunity, that respond to common
antigens on the cell surfaces of potential pathogens. During the past two decades, the
molecular structures and functions of various defense components that participated in
innate immune systems have been well studied in Arthropoda, such as, insects, the
horseshoe crab, freshwater crayfish, and the protochordata ascidian. These defense
molecules include phenoloxidases, clotting factors, complement factors, lectins, protease
inhibitors, antimicrobial peptides, Toll receptors, and other humoral factors found mainly
in hemolymph plasma and hemocytes. These components, which together compose the
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innate immune system, defend invertebrate from invading bacterial, fungal, and viral
pathogens. The innate immune system is the first line of inducible host defense against
bacterial, fungal, and viral pathogens (Hoebe et al., 2004). This defense system is
essential for the survival and perpetuation of all multi cellular organisms (Hoffmann et
al., 1999; Salzet 2001). Invertebrates, which do not possess immunoglobulins, have
developed unique modalities to detect and respond to microbial surface antigens like
lipopolysaccharides (LPS), lipoteichoic acids, lipoproteins, peptidoglycan (PGN) and
(1→3) β-D-glucans. Because both invertebrates and vertebrates respond to these
substances, it is likely that a system recognizing these epitopes emerged at an early stage
in the evolution of animals (Medzhitov and Janeway, 2000; Aderem and Ulevitch, 2000).
Moreover, it is well known that various microbial cell wall components elicit a variety of
responses that depend on species and cell type (Hoffmann et al., 1999; Cooper et al.,
2002).
In invertebrates, toll-like receptor-mediated antimicrobial peptide production
(Lemaitre et al., 1996; Krutzik et al., 2001; Underhill and Orinsky, 2002), hemolymph
coagulation (Iwanaga et al., 1978), melanin formation (Sugumaran, 2002), and lectinmediated complement activation are prominent immune responses. In addition to these
enzyme cascades, a variety of agglutinin-lectins and reactive oxygenproducing and
phagocytic systems cooperate with immune reactions to kill invading pathogens (Bogdan
et al., 2000).
Hemolymph and circulating hemocytes
The hemolymph plasma of crustaceans contains many soluble defense molecules,
such as hemocyanins, various lectins, and C-reactive proteins, and thioester bond
containing proteins. It was evident that large numbers of granular hemocytes
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(amebocytes), which undergo a rapid degranulation on contact with pathogens (Iwanaga
et al., 1998). Hemocytes, which compose more than 99% of circulating cells, contain a
variety of defense molecules, which are located in two types of secretary granules large
(L)-and small (S)-granules (Muta and Iwanaga, 1996a; Iwanaga and Kawabata, 1998).
L-granules selectively store more than 25 defense components . These include clotting
factors, a clottable protein coagulogen, proteinase inhibitors, lectins, and antimicrobial
proteins. In contrast, the S-granules contain at least six antimicrobial peptides and several
proteins . These peptides include large amounts of hairpin-like tachyplesin, tachystatins,
tachycitins and big defensins, which are highly active against Gram-negative and positive bacteria and fungi (Iwanaga et al., 1994b; Iwanaga, 2002). Moreover, circulating
hemocytes are extremely sensitive to bacterial LPS, and respond by degranulating a
number of granular components after LPS-mediated stimulation, which results in the
formation of hemolymph clot. This rapid clotting response is believed to be important for
the animal’s host defense, which involves engulfing of invading microbes, and in
addition prevents hemolymph leakage (Muta and Iwanaga, 1996a). The hemolymphclotting phenomenon was first identified as a prominent defense system in the horseshoe
crab (Limulus polyphemus) (Bang, 1956). When Gram-negative bacteria invade the
hemolymph, hemocytes detect LPS molecules on their surfaces (Ariki et al., 2004), and
then release, via rapid exocytosis, the contents of L- and S-granules (Iwanaga, 1993a,b).
These released granular components include two biosensors, named factors C and G .
These two factors are serine protease zymogens and are autocatalytically activated by
LPS or (1→3)-β-D-glucan, which are major components of the cell walls of Gramnegative bacteria and fungi, respectively. The prophenoloxidase activation system found
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in crustaceans (Ashida and Brey, 1998) is an important aspect of innate immunity, and
functions to detect and kill invading pathogens and to produce melanin and its derivatives
to encapsulate invaders and facilitate wound healing . Hemocyanin of the tarantula
Eurypelma californicum expresses phenoloxidase activity after limited proteolysis with
trypsin and α-chymotrypsin (Decker and Tuczek, 2000). Furthermore, hemocyanins of
the crab Carcinus maenas and the lobster Homarus Americanus also express significant
phenoloxidase activity in the presence of perclorate. Moreover, the phenoloxidase
activities of the hemocyanins of the horseshoe crab Limulus polyphemus (Nellaiappan
and Sugumaran, 1996) and T. tridentatus are induced in the presence of sodium
dodecylsulfate and phosphatidylethanolamine (Sugumaran, 2002). The clotting cascade
of T.tridentatus is linked to prophenoloxidase activation, with the oxygen carrier
hemocyanin functioning as a prophenoloxidase substitute. Active clotting enzyme or
factor B (active form) functionally transform hemocyanin to phenoloxidase, and this
conversion plateaus at a stoichiometry of 1 : 1 without proteolytic cleavage. Interestingly,
proclotting enzyme also induces hemocyanin based phenoloxidase activity, but neither
factors C nor G zymogens have any effect on hemocyanin. Although the functional roles
of these links between the clotting cascade and hemocyanin-based phenoloxidase systems
in vivo is not clear, these suggest that hemocyanin exists abundantly in hemolymph
plasma and that it may participate in the innate immune system of the crustaceans (Nagai
et al.,2001).
Lectin-agglutinin system
The innate immune system of horseshoe crab recognizes invading pathogens by
using a combinatorial method involving lectin-agglutinis with different specificities
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components exposed on the surfaces of pathogens (Crouch et al., 2000; Chiou et al.,
2000; Kawabata et al., 2001). Five types of lectins (tachylectins(TL)-1 to 5) have been
identified in circulating hemocytes and hemolymph plasma (Kawabata and Iwanaga,
1999; Kawabata and Tsuda, 2002). These components function synergistically to form an
effective host defense against invading microbes and foreign substances. Of these
agglutin-lectins, hemocyte-derived TL-1 interacts with Gram-negative bacteria probably
via 2-keto-3-deoxyoctonate (KDO), a constituent of bacterial LPS, and was found to
cause the agglutination. TL-1 is a single-chain protein of 221 amino acid residues with
no N-linked sugar chain, and contains three intra-chain disulfide bonds and a free
cysteine residue. The six tandem repeats of TL-1 are an outstanding structural feature.
TL-1 exists as a monomer in solution, and the 3D structure of TL-1 is dominated by six
β-sheets, which correspond to the six tandem repeats. TL-1 has a six-bladed propeller
structure with a central zinc atom coordinated with three aspartate residues, two serine
residues and one molecule of water. The center of TL-1 also contains a cluster of six
lysine residues one in each of the six repeated structures, which form a hole allowing
interactions with trisaccharides containing KDO (Saito et al., 1995; Kawabata and
Iwanaga, 1999).
The most interesting feature of the 236 amino acid TL-2 sequence is the five tandem
47 amino acid repeats. Moreover, TL-2 does not contain cysteine or N- or O-linked
sugars, and is present as a monomer in solution. TL-2 has a five-bladed β-propeller
structure and its single chain is organized into five sheets, arranged in consecutive order
with five-fold rotational symmetry around a central tunnel (Beisel et al., 1999). The
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structure contains five equivalent binding sites, with virtually identical occupancies and
geometries in the crystal.
The agglutinating activity of TL-3 is equivalent to that of TL-2, but its activity is more
strongly inhibited by the S-type LPSs of several Gram-negative bacteria, but not by the
corresponding R-type LPSs, which indicates that TL-3 has high specificity against Oantigens. Moreover, TL-3 contains 123 amino acid residues in the form of two repeating
sequences and is present as a dimmer in solution (Saito et al., 1997).
TL-4 is a 470kDa 232 amino acid oligomeric glycoprotein (Inamori et al., 1999), and
its agglutinating activity is more potent than those of TL-2 and TL-3. TL-4 is strongly
inhibited by bacterial S-type LPS, but is not inhibited by R-type LPS, which lacks Oantigen.
Of the five types of plasma-derived tachylectins, TL-5A and TL-5B have greatest
hemolymph agglutinating activities. Both TL-5A and TL-5B specifically recognize acetyl
group-containing substances including noncarbohydrates; only the acetyl group is
required for recognition. They strongly agglutinate Gram-negative and-positive bacteria.
The overall sequence identity between the 269 amino acid residues of TL-5A and the 289
residues of TL-5B is 45%, and they share sequence similarity with the COOH-terminal
fibrinogen like globular domain. Interestingly, a collagenous domain in ficolins is
missing in the corresponding regions of TLs-5A and -5B. Thus, since ficolin-like
mannan-binding lectin is known to participate in a novel lectin pathway of the
complement system (Matsushita and Fujita, 2001; Lindahl et al., 2000).
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C-reactive protein
The hemolymph of crustaceans contains another class of bacterial agglutinins,
which is structurally related to mammalian CRP (Iwaki et al., 1999). Limulin, a sialic
acidand phosphorylethanolamine-binding hemagglutinin in the hemolymph plasma of L.
polyphemus, is a hemolytic CRP . Three types of CRPs have been purified from the
plasma of T. tridentatus. These CRPs are named T. tridentatus CRP-1 (tCRP-1), tCRP-2,
and tCRP-3, each of which consists of several isoproteins. tCRP-1 is the most abundant
CRP, and exhibits highest affinity for phosphorylethanolamine-protein conjugate but
lacks both sialic acid-binding and hemolytic activities. tCRP-2 exhibits a higher affinity
to colominic acid, a bacterial polysialic acid, though tCRP-3 shows stronger hemolytic,
sialic acid-binding and hemagglutinating activities than tCRP-2.However, tCRP-3 has no
affinity for colominic acid. Thus, tCRP-3 is a novel hemolytic CRP, which lacks the
common characteristic of CRPs. Twenty-two clones of tCRPs with different deduced
amino acid sequences showing high levels of molecular diversity have been identified
(Iwaki et al., 1999). Moreover, of these tCRP clones only tCRP-3 contains a unique
hydrophobic nonapeptide sequence that appears in the transmembrane domain of a major
histocompatibility complex class I heavy chain of rainbow trout, which suggests that their
hydrophobic nonapeptide patch is important for the hemolytic activity of tCRP-3. The
structural and functional diversities of tCRPs offer an interesting model for the study of
invertebrate innate immunity, which allows survival without the benefit of acquired
immunity.
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Tachyplesin
The S-granules of T. tridentatus hemocytes contain a family of arthropodous
peptide antibiotics, called the “tachyplesin” family, in addition to tachycitins and
tachystatins. The S-granulederived peptides antibiotics bind chitin, but no other
polysaccharides, e.g., cellulose, mannan, xylan, or laminarin. One of these antimicrobial
peptides , tachyplesin, which consists of 17 amino acid residues, was found to
significantly inhibit the growth of Gram-negative and - positive bacteria and fungi
(Iwanaga et al., 1998; Morvan et al., 1997). Tachyplesin has a rigid hairpin loop that is
constrained by two disulfide bridges and adopts an antiparallel β-sheet conformation
connected to a β-turn. In its planar conformation, the five hydrophobic side chains are
localized on one face, and the six cationic side chains on another. Thus, the amphiphilic
structure of tachyplesin is presumed to be closely associated with its antibacterial
activity.
Tachystatins
Tachystatins A, B, and C, consist of 41-44 residues, and exhibit broad-spectrum
antimicrobial activity against fungi and Gram-negative and -positive bacteria (Osaki
et al., 1999). Of these three, tachystatin C is the most effective, and tachystatin A is
homologous to tachystatin B. Tachystatin C contains the same disulfide motif found in
tachystatin A, but shares low sequence similarity with tachystatin A. Tachystatins cause
morphologic changes in budding yeast, and tachystatin C has strong cell lysis activity.
Furthermore, tachystatin C, but not A and B, has hemolytic activity.
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Tachycitin
Tachycitin consists of 73 amino acid residues that contain five disulfide bonds each
with no N-linked sugar (Kawabata et al., 1996). Moreover, although its antimicrobial
activity is only moderate, tachycitin synergistically enhances the antimicrobial activity of
big defensin. The 3D structure of tachycitin is largely composed of NH2- and COOHterminal domains (Suetake et al., 2000). Like tachyplesin, tachycitin also has strong
chitin-binding ability, and contains a similar hairpin loop in a two-stranded β-sheet. In
both tachycitin and tachyplesin hydrophobic residues clustered on the one face of the βhairpin loops probably function as the chitin-binding site. Chitin is a component of the
cell wall of fungi, and is also a major structural component of arthropod exoskeletons.
Thus, the antimicrobial substances probably recognize chitin exposed at the site of a
lesion, and appear to function in wound healing and as antibacterial molecules; in
addition, they may stimulate and accelerate biosynthesis of chitin at sites of injury.
The pro-PO activating system
The pro-PO system consists of several proteins that participate in melanin formation,
cytotoxic reactions, cell adhesion, encapsulation, and phagocytosis, in invertebrates, such
as, ascidians, mollusks, echinoderms, bivalves, and brachiopods (Söderhäll et al., 1994;
Cannon et al. 2004). It is an efficient non-self recognition system and is initiated by the
recognition of the LPSs or peptidoglycans of bacteria and (1 →3)-β-D-glucans of fungi.
The pro-PO system of P. leniusculus, is composed of a protease cascade composed of a
pattern of recognition proteins, several serine protease, their zymogens, and pro-PO. The
active form of pro-PO, phenoloxidase, also known as tyrosinase, catalyzes two successive
reactions; the first is the hydroxylation of a monophenol to an O-diphenol, and the second
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is the oxidation of O-diphenol to O-quinone (Sugumaran, 2002). The production of toxic
quinone intermediates and O-quinones by phenoloxidase is an initial step in the
biochemical cascade of melanin biosynthesis, and is also important in cuticular
scleotization, wound healing, and in the encapsulation of foreign materials for host
defense (Cerenius and Söderhäll, 2002).The pro-PO is synthesized and localized in
hemocyte granules and then released into plasma by exocytosis triggered by pattern
recognition proteins. The LPS binding proteins (Cerenius et al., 1994), are the triggering
molecules of the crayfish pro-PO system, since they bind microbial cell wall components
and induce the activation of serine protease zymogens in the pro-PO system (Lee et al.,
2000). The serine proteases of the crayfish pro-PO system contain one “clip domain”,
which shows homology with horseshoe crab derived big defensin and mammalian βdefensin . This “clip domain” is cleaved upon activation by upstream proteases (Wang
et al., 2001). Moreover, the cleaved “clip domain” in crayfish shows antibacterial activity
in vivo against Gram-negative bacteria, which suggests that the pro-PO activating serine
proteases have a dual function.
Plasma clotting protein
.
As described earlier, vertebrates and invertebrates have separately evolved efficient
molecular mechanisms to immediately form blood clots (Iwanaga, 1993a; Theopold
et al., 2004), which is essential to prevent blood/hemolymph loss in case of injury.
Vertebrates have similar clotting systems, which result in the proteolytically induced
aggregation of fibrinogen into insoluble fibrin. These noncovalently-associated fibrin
aggregates are further stabilized by the formation of intermolecular covalent cross-links
in the presence of factor XIIIa (transglutaminase) and Ca2+. Ca2+-dependent factor
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XIIIa catalyses covalent cross-linking to form an ε-(γ-glutamyl) lysine between the side
chains of specific lysine and glutamine residues on certain proteins (Tokunaga and
Iwanaga, 1993). In the crayfish, hemolymph clotting is based on the direct
transglutaminasemediated cross-linking of a specific plasma protein, which is
homologous to the vitellogenins (Hall et al., 1995). Freshwater crayfish clotting protein is
a lipoprotein that consists of 1,721 amino acids (210 kDa × 2, Table 3) and shares limited
sequence similarity with other lipoproteins, such as mammalian apolipoprotein B and
microsomal triglyceride transfer protein. It also contains a stretch with similarity to the D
domain of von Willebrand factor (Hall et al., 1999). Each of its 210 kDa subunits has
lysine and glutamine side chains, which are covalently cross-linked to each other by
transglutaminase.released from hemocytes or tissues, and starts cross-linking plasmaderived clotting protein in the presence of Ca2+. Moreover, crayfish clotting protein
exists in both sexes, unlike the female-specific vitellogenins. In the presence of an
endogenous transglutaminase, clotting protein molecules rapidly assemble into long and
flexible chains that occasionally branch, as evidenced by electron microscopy. Despite its
similar functions, clotting protein appears not to share any structural similarities with
mammalian fibrinogen or horseshoe crab coagulogen. This indicates that crayfish clotting
protein and lobster fibrinogen, which share sequence similarity with the vitellogenins,
constitute a separate group of clotting factors. Thus, the crustacean clotting proteins are a
second type of gel-forming proteins and are evolutionary related to vitellogenins, but they
should not be considered as true vitellogenins, since they have completely different
functions and are constitutively expressed in both sexes.
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Proteins involved in shrimp immuno-defence mechanisms.
Agglutinins
Similar to other crustaceans, penaeid shrimp have molecules, cells and systems
involved in defensive mechanisms to prevent invasions by microorganisms. In shrimp,
two kinds of proteins are involved in the recognition of microbial products, and their
activation of cellular functions has been described (Vargas-Albores, 1995). The first
group is constitutes multivalent sugar-binding agglutinins, also named hemagglutinins or
lectins. The second group constitutes molecules that are apparently monovalent and do
not induce agglutination, even though they are able to bind sugar residues. Agglutinating
activities have been detected in plasma of Penaeus monodon (Ratanapo & Chulavatnatol
1990), Penaeus stylirostris (Vargas-Albores et al., 1992), Penaeus californiensis
(Vargas-Albores et al. 1993a), P. japonicus (Bacheré et al., 1995) and Penaeus indicus
(Maheswari et al., 1997). However, only the agglutinins from P. monodon and P.
californiensis have been purified and their main properties studied. From P. monodon
plasma, a 420-kDa glycoprotein formed by identical 27-kDa subunits was isolated
(Ratanapo & Chulavatnatol, 1990). This lectin, named monodin, can react with NANA
(N-acetyl neuraminic acid) and other N-acetyl amino sugars as determined by inhibition
studies. Monodin induces the agglutination of Vibrio vulnificus, a major infective
bacterium for prawns, and this agglutination can be specifically inhibited by NANA
(Ratanapo & Chulavatnatol 1992). Similarly, the agglutinin isolated from P.
californiensis plasma can be inhibited by monosaccharides (GalNAc, GlcNAc, NANA)
and glycoproteins (fetuin, submaxillary bovine mucin). Although specificity has not been
completely defined, this agglutinin can react with bacterial LPS and is also capable of
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agglutinating several Vibrios, including V. parahaemolyticus. Furthermore, the reaction
can be inhibited by LPS (Vargas- Albores et al., 1993a). This shrimp LPS-binding
agglutinin (LPS-BA) has an apparent molecular weight of 180-170 kDa and is formed of
four 41-kDa subunits.
Beta glucan binding protein
The second recognizing protein detected in shrimp plasma has the capability to react
with beta glucan, and therefore it is named beta glucan binding protein . BGBP has been
purified from P. californiensis, P. vannamei and P. stylirostris plasma as a 100-kDa
monomeric protein. The amino acid content of these shrimp BGBPs is nearly identical to
the homologues from the freshwater crayfish, P. leniusculus. Major differences could be
noted when comparing BGBP from crustaceans and insects, including differences in
molecular masses and amino acid composition.
Even though amino acid sequence
comparison among BGBP can only be done with the N-terminus of the protein, the
sequence appears to be highly conserved between shrimp and crayfish . There are only 3
different residues between white and yellowlegs shrimp BGBPs and notably, all are
substitutions by conserved amino acids. Whether this region corresponds to a specific
domain important for protein function or is representative of the entire protein
conservation, remains to be analysed once the complete sequence of more BGBPs are
obtained. Although these kinds of proteins have also been found in insects (Ochiai &
Ashida 1988), molecular masses and amino acid compositions are different to shrimp
BGBP (Vargas-Albores et al., 1996). Unfortunately, studies on other beta glucan binding
proteins that stimulate the proPO system are not yet available.
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Prophenol Oxidase system (ProPO)
In shrimp, like in all crustaceans, melanization is due to the action of phenoloxidase (PO)
which promotes hydroxylation of phenols and oxidation of o-phenols to quinones, in
response to foreign matter invading the hemocoele and during wound healing (Ashida &
Yamazaki, 1990; Johansson & Söderhäll, 1989; Söderhäll, 1992; Söderhäll et al., 1994).
These quinones are subsequently transformed to melanin by non-enzymatic reactions.
Although a direct antimicrobial activity has been described for melanin, the production of
reactive oxygen species such as superoxide anions and hydroxyl radicals during the
generation of quinoids (Nappi et al., 1995; Song & Hsieh, 1994) has also an important
antimicrobial role. In addition, biological reactions like phagocytosis, encapsulation and
nodulation are also activated.
Crustacean PO is located inside hemocytic granules as an inactive pro-enzyme called
prophenoloxidase (proPO) and its transformation from proPO to PO involves several
reactions known as the proPO activating system. As in other crustaceans (Ashida et al.,
1983, Smith & Söderhäll 1983; Söderhäll & Hall 1984; Söderhäll & Unestam 1979), the
shrimp proPO system is specifically activated by β-1,3- glucans (Vargas-Albores 1995;
Vargas-Albores et al., 1996; Vargas-Albores et al., 1997) and LPS (Gollas-Galván et al.,
1997; Hernández-López et al., 1996). Therefore, the crustacean proPO system has been
considered as a recognition system (Ashida et al., 1983; Ashida & Yamazaki 1990,
Johansson & Söderhäll 1989; Söderhäll, 1992; Söderhäll et al., 1994). In addition, the
proPO system has been proposed as the invertebrate counterpart of the vertebrate
complement system since it can be activated by β-1,3- glucans (Ashida, 1983; Smith &
Söderhäll, 1983; Vargas-Albores et al., 1993b), has a cascade reaction, and involves
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proteinases (Aspán et al., 1990a; Söderhäll, 1992; Söderhäll et al., 1994). However, other
than these similarities, a direct lytic activity by the proPO systemhas not been detected.
The proposed proPO activation model for crustaceans (Johansson & Söderhäll, 1989;
Söderhäll 1992; Söderhäll et al., 1994) involves a proteolytic cleavage mediated by a
serine-proteinase (Aspan et al., 1990a), namely proPO activating enzyme (PPAE).
Although inshrimp, PPAE has been not purified, its presence and participation on proPO
activation has been detected. Shrimp PPAE is contained as a zymogen inside the
hemocyte granules, together with proPO and is also released during microbial Stimulus.
The activation of PPAE is Ca2+-dependent and the active enzyme can be inhibited by
either melittin or soybean trypsin inhibitor (STI).
In shrimp, the activation of proPO involves in two steps. The first one is the
degranulation that occurs when hemocytes are stimulated by bacteria, LPS or ß-glucans,
and inactive forms of both proPO and PPAE are released. The second one requires the
participation of Ca2+ for the conversion of inactive PPAE to an active proteinase that, in
turn, transforms proPO to active PO (Gollas-Galván et al., 1997). Thus, under in vivo
conditions, PPAE is activated by plasmatic Ca2+ after hemocyte degranulation, which is
induced by externalstimuli (Gollas-Galván et al., 1997), like LPS and ß-glucans.
The coagulation system
Another system activated by microbial products is coagulation. This is an essential
defence response for crustaceans because it prevents both loss of hemolymph through
breaks in the exoskeleton and the dissemination of bacteria throughout the body (Martin
et al., 1991). Shrimp posses type C coagulation (i.e., there is a requirement for plasma
proteins and cellular components) (Tait, 1911). The key plasma protein which constitutes
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the clot has been named clotting protein or CP. It appears to be present in relatively high
concentrations in hemolymph.
CP has been purified and characterized from many other
crustaceans. In all cases, CP was reported as a lipoglycoprotein with a molecular mass
about 420 kDa, and with two identical subunits linked by disulfide bridges. In addition,
the amino acid composition of the white shrimp CP is similar to its homologues from
crayfish (Kopácek et al., 1993), lobster (Fuller& Doolittle, 1971a) and sand crab
(Madaras et al. 1981), even though the latter species has a different type of coagulation
(Ghidalia et al., 1981).
Apparently, cleavage of the N-terminus is not involved in the crustacean event, since
the same N-terminal sequence is present before and after gelation (Fuller& Doolittle
1971b).However, the basis of coagulation is a cellular transglutaminase (TGase) supplied
by hemocytes (probably hyaline cells) under external stimulus. The TGase-catalyzed
reaction results in the formation of intermolecular ε-(γ-glutamyl)-lysine cross-links
between the side chain of a glutamine residue on one polypeptide and the side chain of a
lysine residue on another polypeptide (Lorand & Conrad, 1984).
Immunostimulants as alternative strategy for shrimp disease management
Diseases are still a major constraint to sustainable aquaculture production, especially
for the farming of invertebrates (Bachere, 2003). In intensive systems, fish and shellfish
species are often exposed to stressful conditions, eventually becoming more susceptible
to microbial infections, especially in their larval stages (Smith et al., 2003). In order to
protect them, it is common practice to disinfect the water before use and to apply
chemotherapy (e.g. antibiotics) (Vadstein, 1997). Yet such practices are undesirable since
they promote the selection and dissemination of antibiotic-resistant bacteria in both the
23
target organisms, as well as in the environment (Smith et al., 2003). Nowadays, the use of
preventive approaches, essential for further development of more sustainable aquaculture
practices, are becoming increasingly important, e.g. vaccines, immunostimulants and
probiotics. Contrary to the vertebrates, which have both an innate and a specific immune
system, the defence mechanism in invertebrates depends mainly on a non-specific or
innate immune response to fight infectious diseases (Kurtz and Franz, 2003). The
invertebrate innate immune system presumably recognizes a wide diversity of pathogens,
represented by common molecular patterns (e.g. lipopolysaccharides or peptidoglycans
from bacteria, or β-glucans from bacteria and fungi) rather than structures specific for
particular microbe (Soderhall et al., 1996). In cases where disease outbreaks are cyclic
and can be predicted, immunostimulants may be used to elevate the non-specific defense
mechanisms, to reduce stress and mortalities, and to maintain the health of cultured
organism (Raa, 2000). Although the exact mechanism is still not yet completely
understood, several immunostimulants are being applied in vertebrate and invertebrate
cultures, to induce and build up protection against a wide range of diseases.
The
perceived benefits of immunostimulants are many and varied. Initially immunostimulants
are often naturally occurring molecules that can be obtained from a natural source in
large amounts, e.g. glucans from yeast (Anderson, 1997; Figueras and Santarem,1998;
Bagni et al., 2000; Efthimiou, 1996; Figueras, 1997; Santarem et al., 1997) or chitosan
(Anderson,1997; Anderson and Siwicki,1994; Bullock et al., 2000; Siwicki et al.,1994)
from arthropods such as shrimps shell meal. Potentially they can make cost effective
dietary supplements due to the relatively low cost of their source ingredients. The use of
immunostimulants, given as dietary supplements, can improve the innate defense of the
24
animal providing resistance to pathogens during periods of high stress, such as grading,
sea transfer and vaccination (Robertsen, 1999; Conceicao et al., 2004; Bagni et al., 2000;
Efthimiou, 1996; Figueras,1997; Santarem et al., 1997; Siwicki et al.,1994; Kennedy
et al., 1998). The use of immunostimulants in vaccine formulations especially the β-3,
β1-6 glucans, has given very good antibody responses. So they can be used either to
replace oil based adjuvants, without having the adverse side effects that have been for
these types of adjuvants, or in addition to them (Anderson, 1997; Figueras and Santarem,
1998; Kawakami, 1998; Romalde, 1999). Thus the use of immunostimulants has been
widely accepted by fish farmers.
Types and applications of immunostimulants
The outbreak of diseases is a limiting factor in fish culture. At many fish farms and
hatcheries several antibiotics, vaccines, and chemotherapeutic agents as well as some
immunostimulants have been used to prevent viral, bacterial, parasitic, and fungal
diseases. These immunostimulants mainly facilitate the function of phagocytic cells and
increase their bactericidal activities. Several immunostimulants also stimulate the natural
killer cells, complement, lysozyme and antibody responses of fish.
Bacterial derivatives
Lipopolysccharides: LPS (lipopolysaccharide) is a cell wall component of gramnegative bacteria. LPS can stimulate B cell proliferation, and, LPS injection into red sea
bream Pagrus major has enhance macrophage phagocytic activity (Salati et al., 1987).
The injection with LPS also increase macrophage migratory activity of Pleuronectes
platessa (MacArthur et al., 1985). In vitro, LPS stimulates phagocytosis and the
production
of
superoxide
anions
in
25
Atlantic
salmon
macrophages
(Sakai et al.,1995c).Similarly, LPS stimulates the production of macrophage activating
factor in goldfish lymphocytes (Neumann et al., 1995) and the production of interleukin 1
like molecules in catfish monocytes (Clem et al., 1985).
Freund’s complete adjuvant (FCA), a mineral oil adjuvant containing killed
Mycobacterium butyricum, enhances immune responses and increases the efficacy of
vaccination in fish ( Paterson and Fryer, 1974). Injection with FCA increased protection
(450 times) against A. salmonicida challenge in coho salmon, O. kisutch (Olivier
et al., 1985). This increased protection was also seen against A. hydrophila and V. ordalii.
Immunologically, the activation of macrophages, phagocytosis and killing in fish injected
with FCA caused increased resistance to A. salmonicida (Olivier et al., 1986).The
rainbow trout injected with FCA showed increases in respiratory burst, phagocytic and
NK cell activity of leucocytes and protection against V. anguillarum infection (Kajita
et al.,1992a).
Muramyl
dipeptide:
Muramyl
dipeptide
(MDP)(N-acetyl-muramyl-L-alanyl-D-
isoglutamine), derived from Mycobacterium, elicits immunostimulatory effects such as ,
the activation of macrophages, B lymphocytes and alternative pathway of complement .
Coho salmon injected with a mixture of MDP and modified Freund’s incomplete
adjuvant have 47-fold increase in resistance to A. salmonicida (Olivier et al.,1985). The
intraperitoneal injection of rainbow trout with MDP–Lys increased the phagocytic
activities, respiratory burst and migration activities of kidney leucocytes as well as
resistance of the fish to A. salmonicida challenge (Kodama et al.,1993).
Vibrio bacterin: V. anguillarum bacterin is the most successful vaccine for salmonid
fish, and this efficacy is high after administration by injection, oral dosing and immersion
26
methods. The rainbow trout immersed in V. anguillarum bacterin solution increased
protection to Streptococcus sp (Sakai et al.,1995c). The vaccination of rainbow trout with
attenuated V. anguillarum induced protection against A. salmonicida challenge ( Norqvist
et al.,1989). The immunostimulant effects of vibrio bacterins were also effective in
Kuruma prawns Penaeus japonicus (Itami et al.,1989). The prawns injected or immersed
with the formalin-killed vibrio bacterin experienced reduced mortalities during challenge
by Vibrio injection 30 days later. They also have efficacy in black tiger shrimp (Horne
et al.,1995). The migration of hemocytes treated with vibrio bacterin increased, compared
to non-treated controls (Itami et al., 1989) .This fact suggested the immunostimulation of
vibrio bacterin in prawn and shrimp. It is still unknown what components of V.
anguillarum cells stimulate non-specific immune responses.
Chitin and chitosan: Chitin is a polysaccharide forming the principal component of
crustacean and insect exoskeletons and the cell walls of certain fungi. The rainbow trout
injected with chitin stimulated macrophage activities and an increased resistance to V.
anguillarum infection (Sakai et al.,1992). Injection with chitin increased protection
against P. piscicida in yellowtail, which continued until 45 days after treatment
(Kawakami et al., 1998). Chitin did not show adjuvant effects (Kawakami et al., 1998).
Chitosan, de-N-acetylated chitin, also showed immunostimulatory effects. Brook trout
Salvelinus fontinalis, injected or immersed in chitosan solution increased protection
against A. salmonicida infection (Anderson and Siwicki, 1994) as did rainbow trout
administered chitosan orally (Siwicki et al., 1994). Rainbow trout treated with chitosan
by injection or immersion enhanced immunological parameters in the blood such as,
27
potential killing activity, myeloperoxidase and total Ig concentration (Anderson
et al., 1995).
Glucan: The immune stimulatory effects of glucan have been well-studied. The effects
of several types of glucan; e.g. yeast glucan, peptide-glucan β-1,3, glucan (VST), have
been investigated in fish. Yeast glucan is the most extensively studied of these glucans.
Intraperitoneal injection of yeast glucan prepared from cell walls of Saccharomyces
cervesiae into Atlantic salmon resulted in increased resistance to V. anguillarum, V.
salmonicida and Y. ruckeri (Robertsen et al., 1990).Catfish injected with yeast glucan
enhanced resistance to E. ictaluri (Chen and Ainsworth, 1992). Yeast glucan has been
applied by immersion and oral administration methods
Animal extracts
The extracts from some invertebrates have immunostimulatory effects.
Eel
injected with an extract from the marine tunicate, Ecteinascida turbinate (Ete) and a
glucoprotein fraction of water extract (Hde) from abalone, Haliotis discus hannai,
enhanced phagocytosis and increased survival following A. hydrophila challenge (Davis
and Hayasaka, 1984). However, the survival after infection with E. ictaluri decreased in
channel catfish injected with Ete, although immunoenhancement was observed(Stanley
et al., 1995). Rainbow trout injected with Hde also showed enhanced phagocytic and NK
cell activities, and showed increased survival against V. anguillarum infection (Sakai
et al., 1991).
Plant extracts
A large number of plants have been used in traditional medicine for the
treatment and control of several diseases (Duke, 1987). Some of the medicinal plants
28
have been used as the phytogenic basis immunostimulatory preparations. The injection of
Ocimum sanctum extract into Tilapia mossambicus produced a higher neutrophil activity
(Logambal et al., 2000).
Seaweeds
Several
seaweed polysaccharides can stimulate the fish immune system. In
addition to laminaran, a β-glucan obtained from Laminaria hyperborea that increases the
activity of Atlantic salmon macrophages in vitro (Dalmo and Seljelid, 1995), other
seaweed polysaccharides appear to posses interesting immunomodulatory properties. The
alginates, abundant in brown seaweeds, enhance the migration and phagocytic activity of
carp (Cyprinus carpio L.) macrophages (Fujiki et al., 1997) and increase the phagocytic
activity, respiratory burst and cytokine expression in trout peritoneal leucocytes after
intraperitoneal injection (Peddie et al., 2002). Seaweed polysaccharides can also modify
the resistance of fish to diseases, e.g. an alginate obtained from Ascophyllum nodosum
was shown to enhance the survival of juvenile turbot challenged with Vibrio anguillarum
(Skjermo et al., 1995). Carp injected intraperitoneally with a carrageenan obtained from
the red seaweed Chondrus ocellatus were also found to be more resistant to Edwarsiella
tarda and Aeromonas hydrophila than control fish (Fujiki et al., 1997).
Vitamins
Dietary vitamin C is essential for normal growth and for several physiological
functions in most fishes (Halver, 1989). High levels of dietary vitamin C are reported to
increase resistance to Edwardsiella tarda and E. ictaluri infection in channel catfish
(Durve and Lovell, 1982; Li and Lovell, 1985; Liu et al., 1989), to V. anguillarum and
Ichthyophthirius multifiliis in rainbow trout (Navarre and Halver, 1989; Suzuki and
29
Ai, 1989; Wahli et al., 1995) and to A. salmonicida and V. salmonicida in Atlantic
salmon (Erdal et al., 1991; Hardie et al., 1991). Treatment with high doses of vitamin C
increased complement activities in catfish or Atlantic salmon (Li and Lovell 1985;
Hardie et al., 1991). This stimulated macrophage activating factors, followed by
lymphocyte proliferation (Hardie et al., 1993). Thus it is clear that fishes fed with high
doses of vitamin C have protective immune responses.
Hormones
The relationship between neuroendocrine regulation and the immune system has
recently become the subject of intense investigation (Berczi and Nagy, 1987; Kelley,
1989; Gala, 1991; Auernhammer and Strasburger, 1995). It is also known that GH and
PRL directly affect immunocompetent cells_macrophages, lymphocytes and NK cells. In
fish, exogenous growth hormone (GH) has mitogenic activity on lymphocytes and
activates NK cells (Kajita et al., 1992b; Sakai et al., 1996b). The exogenous GH given to
rainbow trout increased the production of superoxide anion in leucocytes (Sakai
et al.,1995d, 1996a). Another one commonly used hormone is the cytokines.They are
polypeptides or glycoproteins which act as modulators in the immune system. The
existence of several cytokines has been reported in fish (Secombes et al.,1996). The
structures of cytokines such as interleukin 2 have already been reported (Tamai
et al., 1992). Several cytokines can be used as immunostimulants.
Levamisole
Levamisole is an anthelminthic used for the treatment of nematode infections in
man and animals. When coho salmon, Oncorhynchus kisutch, injected with levamisole
mixed with modified Freund’s complete adjuvant_ MFCA ,showed increased resistance
30
to A. salmonicida (Olivier et al., 1985) and carp injected with levamisole showed
enhanced phagocytic activity and myeloperoxidase activity in neutrophils, increased
leucocyte numbers and serum lysozyme levels( Siwicki 1987, 1989). Rainbow trout
showed increased protection against Vibrio anguillarum, when injected with levamisole ,
caused by the enhancement of non-specific immune responses such as phagocytic
activity, chemiluminescance responses of leucocytes and NK cell activities
( Kajita et
al.,1990). Concentration of complement also increased in levamisole-injected rainbow
trout. The immunostimulatory effects of oral administration of levamisole increased the
number of leucocytes, lysozyme activities in serum and phagocytic index of phagocytic
cells (Siwicki, 1989). Although the immunostimulating efficacy of levamisole has been
reported in fish, the optimum doses should be considered.
FK-565
FK-565(heptanoyl-y-D-glutamyl-_L.-meso-diaminopimelyl-_D.-alanine)
is
a
peptide related to lactoyl tetrapeptide(FK-156) isolated from cultures of Streptomyces
olivaceogriseus and has been shown to be active against microbial infection in mice
(Mine et al., 1983). The injection of FK-565 in to rainbow trout increased their resistance
to A. salmonicida, following the activation of phagocytic cells (Kitao and Yoshida,
1986).
31