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Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 645
_____________________________ _____________________________
Characterisation of Some Immune Genes in the Black Tiger Shrimp,
Penaeus monodon
BY
KALLAYA SRITUNYALUCKSANA
ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001
Dissertation for the Degree of Doctor of Philosophy in Physiological Mycology presented atUppsala University in 2001.
ABSTRACTSritunyalucksana, K. 2001. Characterisation of Some Immune Genes in the Black Tiger Shrimp,Penaeus monodon. Acta Universitatis Upsaliensis. Comprehensive Summaries of UppsalaDissertations from the Faculty of Science and Technology 645, 45 pp. Uppsala. ISBN 91-554-5087-3.
The molecular mechanisms of the immune system in shrimp, Penaeus monodon, arecompletely unknown, despite its economic importance as an aquaculture species, especially inAsia and Latin America. The genes and their gene products involved in the prophenoloxidaseactivating system, which is considered to be a non-self recognition and defence system in manyinvertebrates, have been isolated and characterised in shrimp. These include a zymogen of thiscascade, prophenoloxidase (proPO); a cell adhesion protein, peroxinectin and a patternrecognition protein, β-1,3-glucan binding protein (GBP). All proteins are synthesised in shrimphemocytes, not in the hepatopancreas. The shrimp proPO cDNA clone has 3,002 bp and containsan open reading frame of 2,121 bp encoding a putative polypeptide of 688 amino acids, with amolecular mass of 78.7 kDa. Comparison of amino acids sequences showed that this shrimpproPO was more closely to that of another crustacean, the freshwater crayfish, Pacifastacusleniusculus, than to insect proPOs.
Upon activation of the proPO system in shrimp, a cell adhesion activity in the hemolymphis generated. Inhibition of adhesion by an antiserum against the crayfish cell adhesion protein,peroxinectin, revealed that the cell adhesion activity detected in shrimp hemolymph might resultfrom a peroxinectin in shrimp. Indeed, a cDNA clone which encoded shrimp peroxinectin wasisolated with an open reading frame of 2,337 bp encoding a putative protein of 778 amino acidsincluding a signal peptide. Two putative integrin-binding motifs (RGD and KGD) are presentsuggesting that integrin is involved in the adhesion activity. The peroxinectin transcript wasslightly reduced in shrimp injected with a β-1,3-glucan or laminarin.
Also found in shrimp hemolymph was a 31 kDa-GBP that could bind to β-1,3-glucanpolymers such as curdlan and zymosan, but not to LPS. The cDNA sequence of shrimp GBPshowed high similarity to that of crayfish LGBP, other insect recognition proteins as well asbacterial and sea urchin glucanases. Shrimp injected with an insoluble β-1,3-glucan, curdlan orheat-killled Vibrio harveyi did not show any significant changes in relevant mRNA levels.
An attempt to knock out the LGBP expression by its exogeneous dsRNA was done in aproliferating blood cell culture from the hematopoietic tissue of crayfish. We found that theexpression of endogeneous LGBP mRNA could be substantially inhibited by incubation ofdsRNA-LGBP in the cell culture. The effect is quick, specific, and also affects the cellbehaviours.
Kallaya Sritunyalucksana, Department of Comparative Physiology, Evolutionary Biology Centre,Uppsala University, Norbyvägen 18A, SE-752 36, Uppsala, Sweden Kallaya Sritunyalucksana 2001ISSNIBSNPrinted in Sweden by University Printers, Ekonomikum, Uppsala 2001
To my parents, my brother-sisters
and Somsak
4
Preface
This thesis is based on the following papers, which will be referred to in the text by their Roman
numerals:
I. Sritunyalucksana, K., Cerenius, L., Söderhäll, K. (1999) Molecular cloning and
characterization of prophenoloxidase in the black tiger shrimp, Penaeus monodon.
Developmental and Comparative Immunology, 23, 179-186.
II. Sritunyalucksana, K., Wongsuebsantati, K., Johansson, M.W., Söderhäll, K. (2001)
Peroxinectin, a cell adhesive protein associated with the proPO system from the black
tiger shrimp, Penaeus monodon. Developmental and Comparative Immunology, 25, 353-
363.
III. Sritunyalucksana, K., Lee, S.Y., Söderhäll, K. (2001) A β-1,3-glucan binding protein
from the black tiger shrimp, Penaeus monodon. Developmental and Comparative
Immunology (in press)
IV. Sritunyalucksana, K., Söderhäll, K., Söderhäll, I. (2001) RNAi in a hematopoietic cell
culture of the freshwater crayfish, Pacifastacus leniusculus. (in manuscript)
Paper I and II are reprinted with the respective publisher's kind permission.
1999 Elsevier Science Ltd
2001 Elsevier Science Ltd
Contents
5
Contents
Abstract ………………………………………………………………………………...
Preface ………………………………………………………………………………….
Table of contents ………………………………………………………………………
Abbreviations ………………………………………………………………………….
Chapter I : Introduction ………………………………………………………………
Aquaculture ……………………………………………………………
Innate Immunity ………………………………………………………
The prophenoloxidase activating system …………………………….
Peroxinectin, an associated factor of the proPO system ……………
Pattern recognition proteins ………………………………………….
RNA interference (RNAi) ……………………………………………..
Chapter II : Results and Discussion ...………………………………………………..
Shrimp proPO …………………………………………………………
Shrimp peroxinectin …………………………………………………..
Shrimp β-1,3-glucan binding protein (GBP) ………………………..
RNAi in crayfish cell culture model ………………………………….
Conclusions ...…………………………………………………………………………..
Acknowledgements ...…………………………………………………………………..
References ……………………………………………………………………………...
2
4
5
6
7
7
8
11
14
18
20
23
23
25
27
28
30
33
35
Characterisation of some immune genes in shrimp
6
Abbreviations
bp base pair
BGBP β-1,3-glucan-binding protein
CCF-1 coelomic fluid cytolytic factor-1
dsRNA double-stranded RNA
GNBP Gram negative bacteria-binding protein
HLS hemocyte lysate supernatant
Ig immunoglobulins
kDa kilodalton
LPS lipopolysaccharides
MBP or MBL mannose-binding protein or mannose-binding lectin
PG peptidoglycans
pg/L picogram/liter
PGRP peptidoglycan-binding protein
proppA prophenoloxidase activating enzyme
PRMs pattern recognition molecules
PRRs or PRPs pattern recognition receptors or pattern recognition proteins
proPO prophenoloxidase
PTGS Post transcriptional-gene silencing
RNAi RNA interference
SCR short consensus repeats
Introduction
7
Chapter I: Introduction
1.1 Aquaculture
The aquaculture of penaeid shrimp has rapidly grown to a major industry, which on a
worldwide basis, provides not only economic income and a high quality food product, but also
employment to hundreds of thousands of skilled and unskilled workers. In Thailand, for example,
shrimp farming and processing is estimated to employ 150,000 people (Rosenberry, 1995). The
producing countries are mainly in Asia and South America. Thailand has been the leading shrimp
producing country since 1991 by having 70,000 hectares of shrimp ponds producing
approximately 3,000 kilograms per hectare (Rosenberry, 1998). In Ecuador, shrimp farming is
the second largest economic activity. The black tiger shrimp, Penaeus monodon, is now the most
widely cultured shrimp species in the world. However, the rapid expanding shrimp industry
started to face problems in 1992. Diseases have emerged as a major constraint to the sustainable
growth of shrimp aquaculture. Many diseases are linked to environmental deterioration and stress
associated with farm intensification. Under poor farming conditions, it is often opportunistic
diseases caused by bacteria, fungi and protozoa that are constantly present in the pond
environment, which cause death of the shrimp. More than 15 viruses have been identified to
cause diseases in shrimp during the past two decades (Bower et al., 1994). The increase of
disease problems that have devastated and continue to threaten production of several species
throughout the world has emphasised the need to develop tools for the rapid recognition and
control of pathogens. Disease prevention is more important than treatment. Studies towards a
better understanding of defence mechanisms in shrimp constitute one approach to overcome
disease problem to be able to optimise culture conditions so that good shrimp health is retained.
Characterisation of some immune genes in shrimp
8
If shrimps are in good health, their immune defences are more efficient and disease outbreaks
will be less frequent. It appears possible to apply existing knowledge about defence mechanisms
from other arthropods such as insect, crayfish and horseshoe crab, to understand that of shrimp.
Finally, an understanding of the defence system in invertebrates including shrimp may provide
evidence of the origin of vertebrate immunity and lead to unifying concepts in immunology.
1.2 Innate immunity
A key feature of innate immunity is the ability to limit the infectious challenge in the
early hours after the infection occurs. It has been suggested that studies of innate immunity will
lead to the discovery of common molecular mechanisms used for host defence in plants,
invertebrates and vertebrates. The recognition of conserved molecular patterns characteristic of
pathogens is a property of the innate immune system, which is instrumental in initiating and
regulating the adaptive immune response (Medzhitov and Janeway, 1997). The target recognition
of innate immunity is the so-called ¨pattern recognition molecules (PRMs)¨ shared among groups
of pathogens. Host organisms have developed the response to these PRMs by a set of receptors
referred to as ¨pattern recognition proteins or receptors (PRPs or PRRs)¨ (Janeway, 1989). These
patterns include the lipopolysaccharides (LPS) of Gram negative bacteria, the glycolipids of
mycobacteria, the lipoteichoic acids of Gram positive bacteria, the mannans of yeasts, the β-1,3-
glucan of fungi and double-stranded RNAs of viruses (Hoffmann et al., 1999).
In insects, the innate defence system has been studied intensively in Drosophila
melanogaster (Hoffmann et al., 1999). Components such as transcription factors, antimicrobial
defensins, and cecropins, binding proteins and putative members of innate immune cascades have
Introduction
9
been isolated by homology cloning, or by the empirical criterion of up-regulation upon immune
challenge. Toll/NFκB pathway is conserved between insects and mammals to activate non-
specific defence mechanisms in both cases. Toll has been shown to induce the synthesis of
antifungal and antibacterial peptides in Drosophila (Lemaitre et al., 1996), while in mammals,
Toll induces signals required for the activation of the adaptive immune response (Medzhitov et
al., 1997). Recent experiments indicated that mammalian Toll-like receptors are critical in LPS-
mediated signalling in association with LPS-binding protein (LBP) and CD14 (Medzhitov et al.,
1997; Rock et al., 1998).
Proteolytic cascades triggered by nonself recognition molecules have major roles in
innate immunity. Examples are the complement cascade in mammals (Volanakis, 1998),
hemolymph coagulation in horseshoe crab (Kawabata et al., 1996) and the phenoloxidase
mediated melanization in crustaceans and insects (Söderhäll, 1982; Söderhäll et al., 1994). The
complement cascade is activated directly (via alternative and lectin pathways) or indirectly (via
classical pathway) by microorganisms and results in their opsonization for phagocytosis,
chemotaxis or lysis by the assembly on their surface of a pore-forming membrane attack complex
(Fearon and Locksley, 1996). The lectin pathway requires mannose-binding protein (MBP)
(Epstein et al., 1996). MBP recognises sugar moeities on microbe surfaces and results in the
activation of the MBL-associated serine proteases, MASP-1 and -2, which in turn activate the C3
convertase (Matsushita and Fujita, 1995). Recent cloning of MASPs in lamprey (Matsushita et
al., 1998) and tunicates (Ji et al., 1997), C3-like molecules from tunicates (Smith et al., 1999) and
sea urchins (Al-Sharif et al., 1998), and related thioester-containing protein (TEP) in Drosophila
melanogaster (Lagueux et al., 2000) and Anopheles gambiae (Levashina et al., 2001) leads to the
Characterisation of some immune genes in shrimp
10
prediction that the lectin pathway of mammalian complement system seems to be ancient. An
earlier link between recognition of microbial molecular patterns, proteolytic cascades and
activation of host defence came from the studies of the clotting cascade in the horseshoe crab,
Limulus polyphemus (Kawabata et al., 1996; Iwanaga et al., 1998). The proteins participating in
the horseshoe crab clotting system all reside in the hemocytes and, upon activation they are
released from the cytoplasmic L-granules into the hemolymph through rapid exocytosis. Gram
negative bacteria and fungi invading the horseshoe crab hemolymph activate factor C and factor
G, respectively, which results in the formation of an insoluble coagulin gel that limits the
infection (Kawabata et al., 1996; Iwanaga et al., 1998). Factor C in this cascade has five short
consensus repeats (SCR, also called CCP or the sushi domain) (Muta et al., 1991) that are found
in mammalian complement proteins, suggesting an early common origin of the complement and
coagulation cascades. The prophenoloxidase activating system (the proPO system) is an
enzymatic cascade reported in many invertebrates and large amount of information about this
system has come from work done on crustaceans; the freshwater crayfish, Pacifastacus
leniusculus (for reviews see Söderhäll et al., 1994; Söderhäll and Cerenius, 1998). The activation
of the proPO system is brought about by an extremely low amount (pg/L) of microbial cell wall
components such as LPS and β-1,3-glucans. Activation of the proPO system not only leads to the
synthesis of melanin, but also initiates several biological molecules responsible in the defence
system of the crayfish. Recently, Nagai and Kawabata (2000) showed that Tachypleus clotting
enzyme and activated factor B are capable to functionally transform hemocyanin to
phenoloxidase without proteolytic cleavage suggesting that the two host defence systems of
blood coagulation and prophenoloxidase activation are evolutionary related protease cascades.
Introduction
11
1.3 The prophenoloxidase activating system
Invertebrate animals do not have antibodies and therefore have to rely on innate immune
systems, but still they have to be able to recognise foreign materials and respond to it so that
appropriate measures are initiated to combat and destroy invading microorganisms. The ways in
which invertebrate animals recognise and respond to non-self particles or molecules are
beginning to be understood at the molecular level. It has long been recognised that defence
reactions in many invertebrates are often accompanied by melanization. In arthropods, melanin
synthesis is involved in the process of sclerotization, pigmentation and wound healing of the
cuticle as well as in defence reactions. During the formation of melanin, toxic metabolites are
formed which have fungististic activity (Söderhäll and Ajaxon, 1982; St. Leger et al., 1988;
Rowley et al., 1990; Nappi and Vass, 1993). The prophenoloxidase activating system (the proPO
system) is considered to be a non-self recognition and defence system in many invertebrates
(Söderhäll and Cerenius, 1998). The susceptibility of Rhodinus prolixus to Trypanosoma rangeli
infection might be related to the suppression of the activation of proPO in the presence of this
flagellate (Gregorio and Ratcliffe, 1991). Injection of the nonpermissive fungus, Entomophaga
aulicae into lepidopteran insect, Lymantria dispar resulted in an increase of insect phenoloxidase
activity when compared to injection of a permissive strain, E. maimaiga (Bidochka and Hajek,
1998) suggesting that the activation of proPO continues during a brief survival of this fungus in a
nonpermissive host. So far, proPOs have been cloned from 15 invertebrate species, 2 crustaceans
and 13 insects. Several isoforms encoded by different genes have been found in insects (Ashida
and Brey, 1995; Müller et al., 1999), but it is yet not known if they have different functions. The
schematic drawing for the activation of the proPO cascade in crayfish is shown in Figure 1 and
Characterisation of some immune genes in shrimp
12
the molecules involved in this system that have been isolated from the black tiger shrimp,
Penaeus monodon are indicated.
The active enzyme, phenoloxidase (PO; monophenol, L-dopa:oxygen oxidoreductase;
EC1.14.18.1) is responsible for the well-known melanization reaction, which is generally
observed in a wounded area or during an immune response in invertebrates. PO is a bifunctional
copper-containing oxidase catalysing the oxidation of phenolic substance into quinones, which is
further converted to melanin (Sugumaran, 1996). PO has been detected in the hemolymph
(blood) or coelom of both protostomes and deutereostomes, as well as the cuticle of arthropods.
ProPOs have been cloned from many arthropod species, and by comparison of their amino acid
sequences, arthropod proPOs have high similarity to arthropod hemocyanins, but rather remote
relationship to vertebrate tyrosinases. Tyrosinases found in ascidians, Halocynthia roretzi,
resemble vertebrate tyrosinases rather than arthropod proPOs (Sato et al., 1997), since this
enzyme has a signal peptide and a transmembrane domain like vertebrate tyrosinases. PO is
found in different sizes, monomers, homodimers, heterodimers and homotetramers. However, the
subunits from different species fall in the range between 71-83 kDa on a SDS-PAGE gel
(Söderhäll and Cerenius, 1998).
The activation of this proPO cascade is exerted by microbial cell wall components;
lipopolysaccharides (LPS), β-1,3-glucans or peptidoglycans (PG). The recognition of these
nonself molecules is by endogenous PRRs leading to degranulation of hemocytes. Several
components and associated factors of the proPO system have been found to play several
important roles in the defence reaction of the freshwater crayfish (Söderhäll and Cerenius, 1998).
Clotting protein Clot
β-1,3-glucans Lipopolysaccharides (LPS) Peptidoglycans (PG)
β-1,3-glucan binding protein((BGBP)
(Sritunyalucksana et al., submitted)
LPS-binding protein(GNBP)
PG-binding protein (PGRP)
Degranulation Proteins released:Mas-like proteinα2-macroglobulinAntibacterial proteinsKazal inhibitor(Sritunyalucksana et al.,unpublished)
Ca2+
Transglutaminase(Song et al., unpublished)
ppA
Melanin synthesis
Peroxinectin(Sritunyalucksana et al., 2001)
Integrin
EC-SOD
Cell adhesionDegranulation OpsonisationEncapsulationPeroxidase activity
proPO PO
Proteins isolated from shrimp, P. monodon are indicated in bold letters.Figure 1: The prophenoloxidase activating system in crustaceans.
: granules-containing hemocyte
13
(Sritunyalucksana et al., 1999)(Yeh et al., 1999)
Introduction
Characterisation of some immune genes in shrimp
14
Under physiological conditions, arthropod proPOs require a proteolytic cleavage by a specific
protease for activation; the inactive proPO in the freshwater crayfish with a molecular mass of 76
kDa is converted into an active form with a molecular mass of 62 kDa by the prophenoloxidase
activating enzyme (ppA) (Aspán and Söderhäll, 1991; Aspán et al., 1995). A proppA becomes
activated by the presence of PRPs (Aspán et al., 1990). ProppAs cloned from insects and
crustaceans have been shown to be homologous to Tachypleus clotting enzyme, activated factor
B and Drosophila easter (Muta et al., 1990, 1993; Lee et al., 1998; Jiang et al., 1998; Satoh et al.,
1999; Wang et al., 2001). The common feature of arthropod ppA enzymes are that they are serine
proteinases and have clip-like domains (Lee et al., 1998; Wang et al., 2001). The clip-like domain
seems to play several biological functions. The clip-domain of clotting enzyme and factor B in
horseshoe crab is proposed to mediate the functional conversion of hemocyanin to phenoloxidase
(Nagai and Kawabata, 2000). Wang et al. (2001) has recently shown that the recombinant peptide
from the clip-like domain (defensins) of crayfish proppA has an antibacterial activity in vitro.
1.4 Peroxinectin, an associated factor of the proPO system
Several cell adhesion molecules have been discovered and characterised during the past
few years in invertebrates and have shown to participate in immunological processes. These
processes include cell attachment and spreading, nodule formation, encapsulation, agglutination
(or aggregation) and phagocytosis (Johansson, 1999). So far, a few blood cell adhesion molecules
in arthropods have been cloned (Table 1).
Introduction
16
Tab
le 1
: Art
hrop
od b
lood
cel
l adh
esio
n m
olec
ules
S
peci
es
pro
tein
s
cl
oned
R
efer
ence
cell
adhe
sion
R
efer
ence
a
ctiv
ity*
Pac
ifast
acus
leni
uscu
lus
Pero
xine
ctin
Yes
Joha
nsso
n et
al.,
199
5Y
esJo
hans
son
and
Söde
rhäl
l, 19
88
M
asqu
erad
e-lik
e pr
otei
n Y
es
H
uang
et a
l., 2
000
Y
es
Hua
ng e
t al.,
200
0
Pen
aeus
mon
odon
Pe
roxi
nect
in
Yes
Sritu
nyal
ucks
ana
et a
l., 2
001
Yes
1Sr
ituny
aluc
ksan
a et
al.,
200
1
Pen
aeus
pau
lens
is
N
o
Yes
1 Pe
razz
olo
and
Bar
racc
o, 1
997
Lim
ulus
pol
yphe
mus
Lim
unec
tin
Y
es
L
iu e
t al.,
199
1
No
L
imul
us a
gglu
tinat
ion-
ag
greg
atio
n fa
ctor
(L
AF)
Yes
Fujii
et a
l., 1
992
Yes
Fu
jii e
t al.,
199
2
Car
cinu
s m
aenu
s
No
Yes
T
hörn
qvis
t et a
l., 1
994
Bla
beru
s cr
aniif
er
No
Yes
R
anta
mäk
i et a
l., 1
991
Bom
byx
mor
i
Hem
ocyt
in
Y
es
Kot
ani e
t al.,
199
5
Yes
Kot
ani e
t al.,
199
5
Dro
soph
ila m
elan
ogas
ter
C
roqu
emor
t
Yes
Fran
c et
al.,
199
6
Y
es
Fran
c et
al.,
199
6
Pse
udop
lusi
a in
clud
ens
Plas
mat
ocyt
e sp
read
ing
pe
ptid
e (P
SP1)
Yes
Cla
rk e
t al.,
199
8
Yes
2
C
lark
et a
l ., 1
997
* ce
ll ad
hesi
on a
ctiv
ity d
etec
ted
from
pur
ifie
d pr
otei
n.1 c
ell a
dhes
ion
activ
ity d
etec
ted
in h
emol
ymph
2 cel
l adh
esio
n ac
tivity
det
ecte
d in
rec
ombi
nant
pro
tein
exp
ress
ed in
bac
ulov
irus
vec
tor
Characterisation of some immune genes in shrimp
16
Upon activation of the proPO system in crayfish, peroxinectin, a cell adhesion factor
with peroxidase activity is generated (Johansson and Söderhäll, 1988). Crayfish peroxinectin is
synthesized in the blood cells, stored in secretory granules of granular hemocytes in an inactive
form, released in response to stimuli, and activated outside the cells to mediate attachment and
spreading. Besides having cell adhesion and peroxidase activities, crayfish peroxinectin also acts
as a degranulation factor, an encapsulation-promoting factor and an opsonin (for reviews see
Söderhäll and Cerenius, 1994; Sritunyalucksana and Söderhäll, 2000). However, it is important to
emphasize that the peroxidase activity is not a prerequisite for the other biological activities of
peroxinectin (Johanssson et al., 1995). Cross-reactive proteins with similar activities have been
isolated from the insect, Blaberus craniifer (Rantamäki et al., 1991) and from the hemocytes of
the shore crab, Carcinus maenas (Thörnqvist et al., 1994). The sequence of a Drosophila
peroxinectin-related molecule (accession no. AAF78217) has been reported and shows high
similarity to crustacean peroxinectins within the peroxidase domain (Sritunyalucksana et al.,
2001). However, the function of this molecule is yet unknown. Thus, it is suggested that
peroxinectin is widely distributed among arthropod species.
The deduced amino acid sequence of crayfish peroxinectin (Johansson et al., 1995) has
high similarity to both invertebrate and vertebrate peroxidases including human myeloperoxidase
(MPO) (32% identity) (Morishita et al., 1987). Isolated primary human leukocytes and
differentiated myeloid (HL-60) cells have been shown to adhere to MPO, whereas
undifferentiated cells did not (Johansson et al., 1997). Taken together, cell adhesion may thus be
a conserved function of animal peroxidases, in addition to producing a potent microbicidal agent
(Klebanoff, 1991). Crustacean peroxinectin also showed high similarity to Drosophila
Introduction
17
peroxidasin, which is a multidomain protein that combines an enzymatically functional
peroxidase domain with motifs that typically occur as parts of cellular matrix proteins including
four immunoglobulin (Ig) loops and six leucine rich repeats (LRR) (Nelson et al., 1994). The
combination of LRR and Ig loop structures suggests that peroxidasin may mediate adhesion of
cells to the extracellular matrix although this molecule has not yet been shown to exhibit cell
adhesion activity. Thus it is plausible that molecules containing peroxidase domains and having
other biological activities as well as peroxidase activity such as crustacean peroxinectin, human
myeloperoxidase and Drosophila peroxidasin, are likely to be widely distributed amongst animal
species. Recently, a human peroxidasin homologue was found and shown to be up-regulated in
p53-dependent apoptotic cells (Horikoshi et al., 1999).
The adhesive function of peroxinectin is likely to be mediated by the integrin-binding
motifs, KGD- or RGD-motifs (Ruoslahti, 1996). A synthetic peptide derived from the sequence
containing KGD triplet was found to mimic the adhesion activity of the entire protein (Johansson
et al., 1995). Holmblad et al. (1997) reported the presence of an integrin β-subunit on surfaces of
the crayfish hemocytes. Besides binding to integrin, peroxinectin also binds to a peripheral blood
cell surface CuZn-superoxide dismutase (EC-SOD) (Johansson et al., 1999). It was suggested
that peroxinectin might produce hypohalic acid from hydrogen peroxide produced by SOD and as
a consequence, function as an efficient microbicidal attack system to invading microorganisms
(Holmblad and Söderhäll, 1999).
Characterisation of some immune genes in shrimp
18
1.5 Pattern recognition proteins
In invertebrates, an increasing number of so-called ¨pattern recognition proteins (PRPs
or PRRs)¨ (Janeway, 1989) have now been isolated and characterised. These PRPs recognise and
respond to microbial invaders by the presence of signature molecules on the surface of the
intruders. PRRs in mammals, a LPS-binding protein (LBP) (Schumann et al., 1990) and the
cellular receptor CD14 have been well characterised and play roles in stimulating macrophages to
produce cytokines (Medzhitov and Janeway, 1997). Besides microbial cell wall components,
dsRNA has also been reported to behave as PRR (Cella et al., 1999; Chu et al., 1999). DsRNA is
an inducer of type I interferon (IFN) which plays a critical role in antiviral response (Müller et
al., 1994; Manetti et al., 1995) as well as other cytokines including interleukin-6 and -12
(Gendelman et al., 1990; Chu et al., 1999; Verdijk et al., 1999). Cella et al. (1999) showed that
dsRNA as well as viral injection induced the activation and rapid maturation of human dendritic
cells with upregulation of MHC molecules, adhesion and co-stimulatory molecules.
A number of invertebrate pattern recognition proteins (PRPs) have been isolated and
characterised and some of them contain common motifs for example, bacterial glucanase-like
(Lee et al., 1996; Ochiai and Ashida, 2000; Cerenius et al., 1994; Ma and Kanost, 2000; Beschin
et al., 1998; Lee et al., 2000; Kim et al., 2000), bacteriophage lysozyme-like (Yoshida et al.,
1996; Ochiai and Ashida, 1999) and immunoglobulin-like (Sun et al., 1990) motif in their
primary structures. Some of them are lectins that can agglutinate a variety of vertebrate blood
cells (Kopacék et al., 1993; Vargas-Albores et al., 1993). Three molecules isolated from the
coelomic fluid of the earthworm, Eisenia foetida (CCF-1, Beschin et al., 1998), the hemocytes of
crayfish, P. leniusculus (LGBP, Lee et al., 2000), and Drosophila melanogaster (DGNBP1, Kim
Introduction
19
et al., 2000) showed affinity to both β-1,3-glucans and LPS. CCF-1 and LGBP have both been
shown to be involved in the activation of the proPO system. In Drosophila, binding of DGNBP1
to either LPS or β−1,3-glucan induces the synthesis of antimicrobial peptides (Kim et al., 2000).
Recently, it was shown that a masquerade-like protein, a serine protease homologue (Huang et
al., 2000), isolated from P. leniusculus, through proteolytic processing, can bind to LPS, Gram
negative bacteria, and yeast and subsequently participates in bacterial clearance (Lee and
Söderhäll, 2001).
So far, β-1,3-glucan binding proteins (BGBPs) have been cloned from many arthropods;
the horseshoe crab, Tachypleus tridentatus (Seki et al., 1994), the freshwater crayfish, P.
leniusculus (Cerenius et al., 1994), the moth, Manduca sexta (Ma and Kanost, 2000), the
silkworm, Bombyx mori (Ochiai and Ashida, 2000), and the black tiger shrimp, Penaeus
monodon (Sritunyalucksana et al., in press). Although BGBPs have glucanase-like motif, none
has been shown to contain glucanase activity suggesting that the BGBPs developed from a
primitive glucanase and then evolved into proteins without glucanase activity, but instead bind
glucans and after binding, operate as elicitors of defence responses. At present, five Gram-
negative bacterial binding proteins (GNBPs) have been discovered; three in insects, one in the
earthworm and one in a crustacean (Sun et al., 1990; Natori and Kubo, 1996; Dimopoulos et al.,
1997; Lee et al., 1996; Kim et al., 2000; Beschin et al., 1998; Lee et al., 2000). These binding
proteins from insects appear to be functionally similar by having affinity to the Gram-negative
bacterial cell walls and are inducible during injury or infection.
Characterisation of some immune genes in shrimp
20
Insect peptidoglycan recognition protein (PGRP) has been reported to be conserved from
insects to human (Kang et al., 1998). Upon binding to PG, PGRP in Bombyx mori mediates the
activation of the proPO system in the plasma fraction of the silkworm hemolymph and its mRNA
expression is induced upon bacterial challenge (Yoshida et al., 1996; Ochiai and Ashida, 1999).
Insect PGRPs cloned from B. mori and Trichoplusia ni (Kang et al., 1998) are homologous
proteins to bacteriophage lysozyme, although it does not contain the amino acid residues
necessary for catalytic action of the enzyme (Cheng et al., 1994).
1.6 RNA interference (RNAi)
RNA interference (RNAi) is a powerful technique to study gene function in animals,
where in vivo genetic analysis cannot be performed. Double-stranded RNA (dsRNA) is a signal
for gene-specific silencing of expression in a number of organisms (reviews see Montgomery and
Fire, 1998; Fire, 1999; Hunter, 1999; Sharp, 1999). RNAi is considered to be a post-
transcriptional gene silencing process due to that dsRNAs corresponding to exon sequences are
active in RNAi, whereas those corresponding to introns are not (Fire et al., 1998). RNAi is
closely linked to the post-transcriptional gene silencing (PTGS) mechanisms of co-suppression in
plants and quelling in fungi. The ability of dsRNA to promote RNAi in eukaryotic animals is
reminiscent to that of the PTGS in plants where injection of dsRNA can initiate the silencing of
the endogenous gene (Fire et al., 1998; Kennerdell and Carthew, 1998; Lohmann et al., 1999;
Sanchez Alvarado and Newmark, 1999; Wianny and Zernicka-Goetz, 2000).
RNAi is stable, reversible, epigenetic modification triggered by sequence-specific
signals that, in some cases, can spread systemically (Tabara et al., 1998). Biochemical and
Introduction
21
genetic approaches will be needed to unravel the signalling and degradation pathway further as it
could be important in several biological phenomena. The natural function of RNAi and PTGS
appears to be protection of the genome against invasion of mobile genetic elements such as
viruses and transposons, which produce aberrant RNA or dsRNA in the host cells when they
become active. During viral infection in mammals, dsRNAs are produced during its early
replication process. DsRNA is one of pattern recognition molecules reported to be able to activate
the innate immune system. They can induce co-stimulation of T cells (Chu et al., 1999; Hoffman
et al., 1999) and upregulate expression of numerous cytokines including type I interferon (Pestka
et al., 1987; Gendelman et al., 1990; Manetti et al., 1995; Verdijk et al., 1999), which plays a
critical role in antiviral responses (van der Broek et al., 1995; Müller et al., 1994). One of the
proteins that is activated by the presence of dsRNA is protein kinase R (PKR), a serine-threonine
kinase that phophorylates and activates elF2, thereby shutting off protein synthesis (Meurs et al.,
1990), which is an antiviral strategy. The relevance of this recognition system is also underlined
by the fact that many viruses specifically target the dsRNA-binding PKR, to escape immune
recognition (Jacob and Langland, 1996; Katze, 1995). Also, some plant viruses appear to encode
gene products, which block the development of the PTGS state (Kasschau and Carrington, 1998).
RNA-directed RNA polymerase (RdRP), helicase and RNA degrading enzymes (RNases) such as
dsRNases and ssRNases are proposed to be involved in RNAi (Sijen and Kooter, 2000). All plant
species possess RdRP activity although its in vivo function remains unknown. The enzyme is not
needed for virus or viroid replication, but RdRP activity can increase by up to 100 times
following injection with a virus or viroid (Franenkel-Conrat, 1986). Moreover, studies of a
mutant RdRP strain of Arabidopsis was shown to exhibit increased susceptibility to viral
Characterisation of some immune genes in shrimp
22
infection suggested that RdRP might play a role in eliciting an antiviral response. Thus,
restriction of viral infection might be a biological function of the PTGs or RNAi.
Results and Discussion
23
Chapter II: Results and Discussion
In this study, we have isolated and characterised several immune genes and their gene
products associated with the prophenoloxidase activating system in the hemolymph of the black
tiger shrimp, Penaeus monodon. These results contribute to an improved understanding of the
shrimp response to microbial pathogens, a necessary prerequisite for development of rational
strategies to improve health management in aquaculture. The results are summarized and
discussed below.
Paper I: Shrimp proPO
Shrimp proPO cloned from the hemocyte cDNA library shares common characteristics to
other arthropod proPOs cloned so far. By comparison of amino acid sequences by UPGMA
analysis, arthropod proPOs can be classified into two major groups; insect and crustacean
proPOs, respectively. The highly conserved part of its primary sequences is around two copper
binding sites; Cu A and Cu B. In crayfish, it was shown that these sites are active and bind Cu2+
(Aspán et al., 1995). Phenoloxidases and hemocyanins display significant sequence similarity and
the six histidine residues within the two copper binding sites of proPO and hemocyanin are
highly conserved in all arthropod proPOs, including the shrimp proPO. Recently, hemocyanins
from two arthropod species; tarantula, Eurypelma californicum and horseshoe crab, Tachypleus
tridentatus, were shown to exhibit phenoloxidase activity (Decker and Rimke, 1998; Nagai and
Kawabata, 2001).
Characterisation of some immune genes in shrimp
24
It is suggested that hemocyanins may switch to function as phenoloxidase at the site of injury to
prevent microbial invasion or at the growing phase of the animal to harden the exoskeleton after
molting or sclerotization (Decker and Rimke, 1998; Nagai and Kawabata, 2001).
The shrimp proPO has a 3,002 bp cDNA and contains an open reading frame of 2,121 bp
encoding a putative polypeptide with 688 amino acids and with a molecular mass of 78.7 kDa.
Shrimp proPO has no signal peptide as all other invertebrate proPOs except those isolated from
the venom-producing gland of the pupal endoparasitoid wasp, Pimpla hypochondriaca
(Parkinson et al., 2001). It is suggested that proPOs without signal peptide are not secreted by the
endoplasmic reticulum secreting system, but by another process for example cell rupture. Shrimp
proPO has been purified from another penaeid shrimp species, P. californiensis, with a molecular
mass of 114 kDa on SDS-PAGE (Gollas-Galván et al., 1999). The active form with a molecular
mass of 107 kDa was produced after hydrolysis with a commercial proteinase preparation. The
molecular mass of purified proPO from P. californiensis (114 kDa) is quite different compared to
the calculated molecular mass of cloned proPO from P. monodon, which either suggests that
proPO has a post-translational modification process such as glycosylation since glycosylation
sites were found in the shrimp proPO sequence or alternatively, the proPO from P. monodon has
much lower mass than that of P. californiensis, which however seems less likely.
The thioester-like motif present (GCGEQNMI) in the complement components; C3, C4
and α2-macroglobulins was also observed in invertebrate proPOs. In vertebrates, proteolytic
activation of C3 leads to covalent attachment of a C3 cleavage product through a thioester bond
Results and Discussion
25
to the pathogen (Volanakis, 1998). Thioester-containing proteins have been described in several
protostomes and they appear to exhibit α 2- macroglobulin like protease inhibitory activity
(Hergenhahn et al., 1987; Armstrong and Quigley, 1996). Recently, thioester-containing protein-
1 (TEP-1) isolated from Anopheles gambiae was shown to have a function resembles that of
vertebrate complement in promoting phagocytosis (Levashina et al., 2001).
Paper II: Shrimp peroxinectin
Peroxinectin is a multifunctional immune protein first found in crayfish and its activities
are generated concomitant with the activation of the proPO system. In this study, we cloned
peroxinectin from a shrimp hemocyte cDNA library and found that it has an overall similarity to
crayfish peroxinectin as well as peroxidases from vertebrates and invertebrates. The cloned
shrimp peroxinectin contains the conserved six disulfide bridges as well as the amino residues
necessary for the catalytic activity of myeloperoxidase (Zeng and Fenna, 1992) suggesting that
shrimp peroxinectin has a peroxidase activity. This has been shown to be the case for crayfish
peroxinectin (Johansson et al., 1995). The mechanism of how peroxinectin functions in vivo is
still unclear, although two receptors of this molecule have been found. Crayfish peroxinectin can
bind to a peripheral extracellular superoxide dismutase (EC-SOD) suggesting it might also be
involved in the production of hypohalic acid and reactive oxygen intermediates, which are toxic
to the microorganism (Holmblad and Söderhäll, 1999). Shrimp peroxinectin has two integrin-
binding motifs in its sequence suggesting the adhesion might be mediated through an integrin
receptor. An integrin receptor was recently isolated and characterised from the freshwater
crayfish (Holmblad et al., 1997). One of these functions of peroxinectin is to act as an opsonin
Characterisation of some immune genes in shrimp
26
(Thörnqvist et al., 1994). Taken together, it might be possible that binding of peroxinectin to its
integrin receptor results in the proximity of the microorganism to the hemocyte surface and then
the production of toxic subtances can occur via EC-SOD which may result in the destruction of
microorganisms.
The expression of peroxinectin is affected by challenging shrimp by microbial cell wall
components. We showed that the level of peroxinectin transcript was slightly reduced 2 hour-post
laminarin or LPS injection. Most likely, the decrease in peroxinectin transcript is due to the
reduction of the number of peroxinectin-expressing cells. It has been shown that during the early
period of infection in crustaceans, a reduction in hemocyte number is observed (Persson et al.,
1987). It might be possible that the hemocytes collected in this early period is newly synthesised
blood cells with low abundance of peroxinectin transcript.
We also checked the presence of peroxinectin in shrimp hemocyte lysate supernatant by
using an immunoblotting assay. An affinity-purified polyclonal antibody against crayfish
peroxinectin could detect one single band with a molecular mass of approximately 80 kDa. We
also found that either granular cell or semigranular hemocytes of shrimp can mediate cell
adhesion activity in the presence of a HLS in which proPO is in its active form, but not in which
proPO is in its non-active form. Polyclonal antibody prepared against crayfish peroxinectin was
shown to be able to inhibit this cell adhesion activity in shrimp. Clearly, therefore, peroxinectin is
a proPO system-associated protein and seems likely to be present among many crustacean
species.
Results and Discussion
27
PaperIII: Shrimp β-1,3-glucan binding protein (GBP)
Several recognition proteins have been identified in penaeid shrimp, but none of them has
been cloned so far. β–1,3-glucan binding proteins (BGBP) from Penaeus californiensis, P.
stylirostris and P. vannamei were reported (Vargas-Albores et al., 1996, 1997; Yepiz-Plascencia
et al., 1998). They have the same characteristics as that of crayfish BGBP, since it is a 100 kDa
monomeric protein and they show similar amino acid composition and N-terminal sequence to
that of crayfish BGBP (Cerenius et al., 1994). Shrimp BGBP is involved in the activation of the
proPO system and it was found that it is the same protein as LP1, a lipid transport protein found
in another penaeid shrimp, P. semisucultus (Lubzens et al., 1997). In this study, we cloned a
β–1,3-glucan binding protein from a hemocyte cDNA library of P. monodon (Sritunyalucksana et
al., in press). We found that the cloned shrimp GBP has a high sequence similarity to other
invertebrate recognition proteins as well as bacterial glucanases (Hahn et al., 1995). The mature
protein has an estimated molecular mass of 39.5 kDa and a predicted pI of 5.5. The amino acids
necessary for the catalytic activity of bacterial glucanase are conserved in shrimp GBP, but since
none of the invertebrate recognition proteins with glucanase-like domains has been shown to
exhibit glucanase activity, it is likely that shrimp GBP also lacks such activity. Alignment of the
shrimp sequence to other invertebrate recognition proteins reveals a high homology at the N-
terminal region of all sequences suggesting that this region is involved in recognition of
microorganisms, which has been shown to be the case for BGBP of Bombyx mori (Ochiai and
Ashida, 2000). Shrimp GBP mRNA expression is not significantly changed after the injection of
laminarin or heat-killed bacteria, Vibrio harveyi suggesting that this protein is constitutively
expressed. This is in agreement with the studies done in BGBP and LGBP from another
Characterisation of some immune genes in shrimp
28
crustacean, P. leniusculus (unpublished data). In one insect, Manduca sexta, the level of its β-1,3-
glucan recognition protein (GRP) mRNA in fat body did not increase significantly after larvae
were injected with bacteria or yeast (Ma and Kanost, 2000). In contrast, PRPs expression from
other insects, Gram negative bacteria binding protein (GNBP, Lee et al., 1996) and BGBP from
B. mori, GNBP from H. cunea (Shin et al., 1998), and A. gambiae (Dimopoulos et al., 1997) were
all shown to be inducible upon microbial challenges.
We found a β-1,3-glucan-binding protein (GBP) in shrimp by using an immunoblotting
assay. Shrimp GBP has a molecular mass of approximately 31 kDa under reducing and non-
reducing conditions suggesting that there is no disulfide linkage in its native molecule. The
molecular mass determined by SDS-PAGE is lower than the calculated molecular mass of its
cDNA sequence suggesting that this protein is being processed. It could bind to only β-1,3-
glucan such as curdlan and zymosan, but not to LPS indicating that its binding is specific for β-
1,3-glucan. Several other protein bands could bind to the curdlan, but these bands could not be
detected by anti-crayfish LGBP antibody suggesting that there are several molecules in shrimp
HLS including shrimp GBP that can recognise β-1,3-glucans.
Paper IV: RNAi in crayfish cell culture model
DsRNA is proposed to be an active intermediate of the process called ¨RNA interference
(RNAi)¨ in invertebrates and vertebrates, ¨quelling¨ in fungi and ¨post-transcriptional gene
silencing (PTGS) or co-suppression¨ in plants. The mechanism of how dsRNA mediates the
silencing of gene expression is still unclear. In this preliminary study, two defence genes; LGBP
and peroxinectin, were chosen to study the effect of dsRNA in a hematopoietic cell culture
Results and Discussion
29
from crayfish. Söderhäll and Söderhäll (Patent application) have developed for the first time a
proliferating cell culture from a hematopoietic tissue of the a crustacean; the freshwater crayfish,
Pacifastacus leniusculus, which is beneficial to study crustacean immunity. We found that
dsRNA-LGBP could substantially, but not completely, inhibit the expression of the endogeneous
LGBP transcript in the crayfish hematopoietic cell culture system. The results from RT-PCR
showed that the level of LGBP transcript was reduced at day 1 as well as day 3 post dsRNA-
LGBP treatments. The effect of dsRNA is specific, as treatment of the cells with dsRNA-
peroxinectin could not significantly reduce the expression of LGBP transcript. Results from in
situ hybridization revealed that the number of cells stained with a Dig-labelled LGBP fragment
was lower in the cell culture treated with dsRNA-LGBP, than in cells treated with dsRNA-
peroxinectin or cells in buffer control. These results are consistent with the observation that
RNAi leads to reduced mRNA levels in Drosophila S2 cell culture, as measured by in situ
hybridisation and Northern blotting (Hammond et al., 2000; Clemens et al., 2000). We also found
the change in cell behaviour after treatment with dsRNA. The cells treated with dsRNA were
round-shaped and did not spread well compared to the cells in buffer control which usually attach
and spread to the bottom of the well. However, the percentage of the stained cells varied highly
between individual experiment suggesting that in situ hybridisation may not be useful as a
quantitative assay. We also need to improve the transfection efficiency in order to reduce the
amount of dsRNA used in the cell culture system.
Characterisation of some immune genes in shrimp
30
Conclusions
The goal of this study is to hopefully improve the understanding of shrimp defence
system. We have isolated and characterised several immune genes associated with the proPO
system in the hemolymph of the black tiger shrimp, Penaeus monodon and have shown that they
are part of the defence system in shrimp. These genes include prophenoloxidase (proPO),
peroxinectin and β-1,3-glucan binding protein (GBP). The primary structures of these immune
genes from shrimp and another crustacean, the freshwater crayfish, Pacifastacus leniusculus, are
very similar which suggest that the immune defense between these two species are likely to be
very similar. Infection or stress has an effect on the expression of the immune genes as has been
shown in shrimp and other invertebrate species. Thus, next step is to evaluate whether these
genes found in shrimp can be useful as reagents to monitor an immune response, the immune
capacity, or the health status of cultured shrimp.
The mechanism of the proPO system in crustacean immunity comprises three processes;
recognition, activation of proPO and amplification of the system. One protein involved in the
recognition step in shrimp is GBP. It can bind to insoluble glucans such as curdlan and yeast
zymosan A, but not to LPS. Another recognition molecule, a 100 kDa monomeric BGBP, have
been reported in the plasma of crayfish as well as in three penaeid shrimp, P. vannamei, P.
californiensis and P. stylirostris, which suggests that this protein is likely to be present in P.
monodon too. Besides having a function as a PRP, it was found to be involved in the reproductive
system of the animals and is the same protein as LP1 in another penaeid shrimps, P.
semisulcatus. Thus, it appears that the same protein may be involved in both reproduction and
immunity.
Conclusions
31
The terminal reaction of the proPO system is to activate the zymogen proPO into active
PO, which will lead to the production of phenolic radicals that are toxic to microbes, and also to
polymerise into melanin. In crustaceans including shrimp, only one proPO gene has been found,
whereas in insects, several isoforms have been isolated, altthough it is still unknown if they have
different functions. Recently, hemocyanins from tarantula and horseshoe crab are reported to
function as phenoloxidase. It is probable that crustacean hemocyanin also have dual functions as
oxygen transporter and phenoloxidase. The conversion of hemocyanin to phenoloxidase might
occur at the site of infection to prevent the invading microorganisms from entering the hemocoel
or take part in the sclerotization process of the animal.
Shrimp cell adhesion activity has been found when the proPO system is activated
suggesting that it is a proPO system-associated factor. The cloned shrimp peroxinectin was
isolated and found to posses a peroxidase domain with conserved amino residues necessary for
peroxidase activity suggesting that shrimp peroxinectin has peroxidase activity, which has shown
to be the case in that of crayfish. Peroxinectin, is proposed to be involved in the amplification
process of the proPO system. After binding to its receptor, it causes degranulation of hemocytes
and thus amplifies the release of the proPO system. It may also be involved in the production of
hypohalic acids and reactive oxygen intermediates (ROIs), which are toxic to microorganisms, as
it was found to be able to bind to an extracellular superoxide dismutase (EC-SOD).
One way to explore the function of a gene is to knock out the expression of that gene.
DsRNA has been shown to inhibit its cognate mRNA expression both in vivo and in vitro. Its
mechanism is shown to be at the post-transcriptional level. A preliminary study of the effect of
dsRNA in crayfish was done in a hematopoietic cell culture system. For the first time a
Characterisation of some immune genes in shrimp
32
proliferating blood cell culture from hematopoietic tissue has been developed from invertebrates
(Söderhäll and Söderhäll, patent application) which then can be used to study the function of
certain immune genes by employing RNA interference technique. We found that crayfish LGBP
mRNA expression could be substantially, even not completely, inhibited since incubation of
dsRNA-LGBP in the cell culture. The inhibitory effect is specific by the incubation of dsRNA-
peroxinectin in the cell culture could not significantly inhibit the expression of LGBP. The
silencing of LGBP mRNA expression caused changes in cell behaviour and the response is quick
since the effect could be seen from day 1 post dsRNA incubation. Thus, this dsRNA-mediated
gene silencing could be a useful tool to study the function of some immune genes in crayfish, in
which genetic manipulation can not be performed.
Acknowledgements
33
Acknowledgements
This thesis was carried out at the department of Comparative Physiology,
Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden. I would like to thank all
those who has directly and indirectly contributed to this thesis.
Professor Kenneth Söderhäll, my supervisor, for providing me with the opportunity to pursue
my Ph.D. study in Sweden, for good advice on science, constantly guiding me throughout my
study, teaching me how to think scientifically and independently.
My special appreciation is to Professor Timothy W. Flegel for his expert guidance and
extensive suggestions on shrimp aquaculture.
Docent Lage Cerenius, Mats W. Johansson, Martin Hall and Irene Söderhäll for interesting
discussions and sharing scientific interests.
My special thanks to Ragnar Ajaxon and Anbar Khodabandeh for wonderful technical
support.
My former colleagues; Cecilia Lindholm, Tornbjörn Holmblad, Tien-Sheng Huang, Pia
Keyser, Ruigong Wang, Maria Lind and Hans Lindmark.
My wonderful present colleagues; Gunnar Andersson, Per-Ove Thörnqvist, Karin Johansson,
Susan Mayo, So Young Lee, Pikul Jiravanichpaisal, Cristiane de Albuquerque Cavalcanti
Jacobsen and Tove Andrén. Thanks for sincerity, sharing and creating a lovely atmosphere in
the lab.
I express my thanks to my lovely Thai friend, Eakaphun Bangyeekhun, for his help and
friendship that I can always count on.
Also, to all of my Thai and foreign friends in Uppsala for making my almost five years-stay
an invaluable experience.
Characterisation of some immune genes in shrimp
34
I would never be able to make it to this point without the constant trust and support of my
parents, my grandmother and my brother-sisters. I love all of you very much.
Last, but not least, I thank my husband, Somsak Dangtip, for your constant encouragement
and always being there for me. You are my hero.
This work was supported by grants from the Swedish Natural Science Research
Council and the Swedish Council for Forestry and Agricultural Research and Eliassons
Fundation. My first year as a Ph.D. student, I received financial support from BIOTEC,
Thailand.
References
35
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