Characterisation of Some Immune Genes in the Black Tiger - DiVA

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.


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


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


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


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


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).


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.


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


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).


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


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


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.


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


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


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.


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


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.


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

References:

1. Al-Sharif, W.Z., Sunyer, J.O., Lambris, J.D., Smith, L.C. 1998. Sea unchin coelomocytespecifically express a homologue of the complement component C3. J. Immunol. 160:2983-2997.

2. Ashida, M., Brey, P.T. 1995. Role of the integument in insect defense: prophenoloxidasecascade in the cuticular matrix. Proc. Natl. Acad. Sci. USA 92:10698-10702.

3. Armstrong, P.B., Quigley, J.P. 1996. Immune function of α2-macroglobulin in invertebrates.Prog. Mol. Subcell. Biol. 15:101-130.

4. Aspán, A., Huang, T.S., Cerenius, L., Söderhäll, K. 1995. cDNA cloning of prophenoloxidasefrom the freshwater crayfish Pacifastacus leniusculus and its activation. Proc. Natl. Acad. Sci.USA 92:939-943.

5. Aspán, A., Sturtevant, J., Smith, V.J., Söderhäll, K. 1990. Purification and characterization of aprophenoloxidase activating enzyme from crayfish blood cells. Insect Biochem. 20:709-718.

6. Aspán, A., Söderhäll, K. 1991. Purification of phenoloxidase from crayfish blood cells and itsactivation by endogenous serine proteinase. Insect Biochem. 21:363-373.

7. Beschin, A., Bilej, M., Hanssens, F., Raymakers, J., Van Dyck, E., Revets, H., Brys, L.,Gomez, J., De Baetselier, P., Timmermans, M. 1998. Identification and cloning of a glucan- andlipopolysaccharide-binding protein from Eisenia foetida earthworm involved in the activation ofprophenoloxidase cascade. J. Biol. Chem. 273: 24948-24954.

8. Bidochka, M.J., Hajek, A.E. 1998. A nonpermissive entomophthoralean fungal infectionincreases activation of insect prophenoloxidase. J. Invert. Pathol. 72:231-238.

9. Bower, S.M., MacGladdery, S.E., Price, I.M. 1994. Synopsis of infectious diseases andparasites of commercially exploited shellfish. Ann. Rev. Fish Dis. 4:1-201.

10. Cella , M., Salio, M., Sakakibara, Y., Langen, H., Julkunen, I., Lanzavecchia, A. 1999.Maturation, activation and protection of dendritic cells induced by double-stranded RNA. J. Exp.Med. 189:821-829.

11. Cerenius, L., Liang, Z., Duvic, B., Keyser, P., Hellman, U., Palva, E.T., Iwanaga, S.,Söderhäll, K. 1994. A β-D-glucan binding protein in crustacean blood. Structure and biologicalactivity of a fungal recognition protein. J. Biol. Chem. 269:29462-29467.

12. Cheng, X., Zhang, X., Pfugrath, J.W., Studier, F.W. 1994. The structure of bacteriophage T7lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase. Proc. Natl. Acad. Sci. USA91:4034-4038.


36

13. Chu, W.M., Ostertag, D., Li, Z.W., Chang, L., Chen, Y., Hu, Y., Williams, B., Perrault, J.,Karin, M. 1999. JNK2 and IKKβ are required for activating the innate response to viral infection.Immunity 11:721-731.

14. Clark, K.D., Pech, L.L., Strand, M.R. 1997. Isolation and identification of a plasmatocyte-spreading peptide from the hemolymph of the lepidopteran insect Pseudoplusia includens. J.Biol. Chem. 272:23440-23447.

15. Clark, K.D., Witherell, A., Strand, M.R. 1998. Plasmatocyte spreading peptide is encoded byan mRNA differentially expresses in tissues of the moth Pseudoplusia includens. Biochem.Biophys. Res. Commun. 250:479-485.

16. Clemens, J.C., Worby, C.A., Simonson-Leff, N., Muda, M., Maehama, T., Hemmings, B.A.,Dixon, J.E. 2000. Use of double-stranded RNA interference in Drosophila cell lines to dissectsignal transduction pathways. Proc. Natl. Acad. Sci. USA 97:6499-6503.

17. Decker, H., Rimke, T. 1998. Tarantula hemocyanin shows phenoloxidase activity. J. Biol.Chem. 273:25889-25892.

18. Dimopoulos, G., Richman, A., Müller, H.M., Kafatos, F.C. 1997. Molecular immuneresponses of the mosquito Anopheles gambiae to bacteria and malaria parasites. Proc. Natl. Acad.Sci. USA 94:11508-11513.

19. Epstein, J., Eichbaum, O., Sheriff, S., Ezekowitz, R. 1996. The collectins in innate immunity.Curr. Opin. Immunol. 8:29-35

20. Fearon, D.T., Locksley, R.M. 1996. The instructive role of innate immunity in the acquiredimmune response. Science 272:50-54.

21. Fire, A. 1999. RNA-triggered gene silencing. Trends Genet. 15:358-363.

22. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C. 1998. Potent andspecific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature391:806-811.

23. Franc, N.C., Dimarcq, J.L., Lagueux, M., Hoffman, J., Ezekowitz, R.A. 1996. Croquemort, anovel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. Immunity4:431-443.

24. Franenkel-Conrat, H. 1986. RNA-directed RNA polymerases of plants. Crit. Rev. Plant Sci.4:213-226.

References

37

25. Fujii, N., Minetti, C.A.S.A., Nakhasi, H.L., Chen, S.W., Barbehenn, E., Nunes, P.H., Nguyen,N.Y., 1992. Isolation, cDNA cloning, and characterization of an 18-kDa hemagglutinin andamebocyte aggregation factor from Limulus polyphemus. J. Biol. Chem. 267:22452-22459.

26.Gendelman, H.E., Friedman, R.M., Joe, S., Baca, L.M., Turpin, J.A., Dveksler, G., Meltzer,M.S., Dieffenbach, C. 1990. A selective defect of interferon alpha production in humanimmunodefficiency virus-infected monocytes. J. Exp. Med. 172:1433-1442.

27. Gollas-Galván, T., Hernández-Lopéz, J., Vargas-Albores, F. 1999. Prophenoloxidase frombrown shrimp (Penaeus californiensis) hemocytes. Comp. Biochem. Physiol. 122B:77-82. 1999

28. Gregorio, E.A., Ratcliffe, N.A. 1991. The prophenoloxidase system in vitro interaction ofTrypanosoma rangeli with Rhodnius prolixus and Triatoma infestans hemolymph. ParasiteImmunol. 13:551-564.

29. Hahn,M., Keitel, T., Heinemann,U. 1995. Crystal and molecular structure at 0.16-nmresolution of the hybrid Bacillus endo-1,3-1,4-beta-D-glucan-4-glucanohydrolase H (A16-M).Eur. J. Biochem. 232:849-858.

30. Hammond, S.M., Berstein, E., Beach, D., Hannon, G.J., 2000. An RNA-directed nucleasemediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293-296.

31. Hergenhahn, H.G., Hall, M., Söderhäll, K. 1987. Purification and characterization of an α2-macroglobulin-like proteinase inhibitor from plasma of the crayfish Pacifastacus leniusculus.Biochem. J. 255:801-806.

32. Hoffmann, J.A., Kafatos, F.C., Janeway, C.A., Ekekowitz, R.A.B. 1999. Phylogeneticperspectives in innate immunity. Science 284:1313-1318.

33. Holmblad, T., Söderhäll, K. 1999. Cell adhesion molecules and antioxidative enzymes in acrustacean, possible role in immunity. Aquaculture 172:111-123.

34. Holmblad, T., Thörnqvist, P.O., Söderhäll, K., Johansson, M.W. 1997. Identification andcloning of an integrin β subunit from hemocytes of the freshwater crayfish Pacifastacusleniusculus. J. Exp. Zool. 277:255-261.

35. Horikoshi, N., Cong, J., Kley, N., Shenk, T. 1999. Isolation of differentially expressedcDNAs from p53-dependent apoptotic cells; Activation of the human homologue of theDrosophila peroxidasin gene. Biochem. Biophys. Res. Com. 261:864-869.

36. Huang, T.S., Wang, H., Lee, S.Y., Johansson, M.W., Söderhäll, K., Cerenius, L. 2000. A celladhesion protein from the crayfish, Pacifastacus leniusculus, a serine proteinase homologuesimilar to Drosophila masquerade. J. Biol. Chem. 275:9996-10001.


38

37. Hunter, C.P. 1999. Genetics: a touch of elegance with RNAi. Curr. Biol. 17:R440-R442.

38. Iwanaga, S., Kawabata, S.I., Muta, T. 1998. New types of clotting factors and defensemolecules found in horseshoe crab hemolymph: Their structures and functions. J. Biochem(Tokyo) 123:1-15.

39. Jacobs, B.L., Langland, J.O. 1996. When two strands are better than one: the mediators andmodulators of the cellular responses to double-stranded RNA. Virology 219:339-349.

40. Janeway, C.A.Jr. 1989. Approaching the asymptole? Evolution and revolution inimmunology. Cold Spring Habour Symp. Quant. Biol. 54:1-13.

41. Ji, X., Azumi, K., Sasaki, M., Nonaka, M. 1997. Ancient origin of the complement lectinpathway revealed by molecular cloning of mannan binding protein-associated serine proteasefrom urochordate, the Japanese ascidian, Halocynthia roretzi. Proc. Natl. Acad. Sci. USA94:6340-6345.

42. Jiang, H., Wang, Y., Kanost, M.R. 1998. Pro-phenoloxidase activating proteinase from aninsect, Manduca sexta: a bacteria-inducible protein similar to Drosophila easter. Proc. Natl.Acad. Sci. USA 95:12220-12225.

43. Johansson, M.W. 1999. Cell adhesion molecules in invertebrate immunity. Dev. Comp.Immunol. 23:303-315.

44. Johansson, M.W., Holmblad, T., Thörnqvist, P.O., Cammarata, M., Parrinello, N., Söderhäll,K. 1999. A cell-surface superoxide dismutase is a binding protein for peroxinectin, a cell-adhesive peroxidase in crayfish. J. Cell Sci. 112:917-925.

45. Johansson, M.W., Lind, M.I., Holmblad, T., Thörnqvist, P.O, Söderhäll, K. 1995.Peroxinectin, a novel cell adhesion protein from crayfish blood. Biochem. Biophys. Res. Com.216:1079-1087.

46. Johansson, M.W., Patarroyo, M., Öberg, F., Siegbahn, A., Nilsson, K. 1997. Myeloperoxidase

mediates cell adhesion via the αMβ2 integrin (Mac-1, CD11b/CD18). J. Cell Sci. 110:1133-1139.

47. Johansson, M.W., Söderhäll, K. 1988. Isolation and purification of a cell adhesion factor fromcrayfish blood cells. J. Cell Biol. 106:1795-1803.

48. Kang, D., Liu, G., Lundström, A., Gelius, E., Steiner, H. 1998. A peptidoglycan recognitionprotein in innate immunity conserved from insects to humans. Proc. Natl. Acad. Sci. USA.95:10078-10082.

References

39

49. Kasschau, K.D., Carrington, J.C. 1998. A counterdefensive strategy of plant viruses:suppression of posttranscriptional gene silencing. Cell 95:461-470.

50. Katze, M.G. 1995. Regulation of the interferon-induced PKR: can virus cope? TrendsMicrobiol. 3:75-78.

51. Kawabata, S., Muta, T., Iwanaga, S. 1996. Clotting cascade and defense molecules found inthe hemolymph of horseshoe crab. In: New Directions in Invertebrate Immunology, Söderhäll,K., Iwanaga, S., Vasta, G.R. (Eds.), SOS Publications, Fair Haven. pp. 255-284.

52. Kennerdell, J.R., Carthew, R.W. 1998. Use of dsRNA-mediated genetic interference todemonstrate that frizzled and frizzled 2 act in the Wingless pathway. Cell 95:1017-1026.

53. Kim, Y.S., Ryu, J.H., Han, S.J., Choi, K.H., Nam, K.B., Jang, I.H., Lemaitre, B., Brey, P.T.,Lee, W.J. 2000. Gram-negative bacteria-binding protein, a pattern recognition receptor forlipopolysaccharide and β-1,3-glucan that mediates the signaling for the induction of innateimmune genes in Drosophila melanogaster cells. J. Biol. Chem. 275:32721-32727.

54. Klebanoff, S.J. 1991. Myeloperoxidase: occurence and biological function. In: Everse, J.,Everse K.E., Grisham M.B., (Eds.), Peroxidase in chemistry and biology. Boca Raton, FL: CRCPress. pp. 2-35.

55. Kopácek, P., Grubhoffer, L., Söderhäll, K. 1993. Isolation and characterization of ahemagglutinin with affinity for lipopolysaccharides from plasma of the crayfish Pacifastacusleniusculus. Dev. Comp. Immunol. 17:407-418.

56. Kotani, E., Yamakawa, M., Iwamoto, S.I., Tashiro, M., Mori, H., Sumida, M., Matsubara, F.,Taniai, K., Kadona-Okuda, K., Kato, Y., Mori, H. 1995. Cloning and expression of the genehemocytin, an insect humoral lectin with is homologous with the mammalian von Willebrandfactor. Biochim. Biophys. Acta 1260:245-258.

57. Lagueux, M., Perrodou, E., Levashina, E.A., Capovilla, M., Hoffman, J.A. 2000. Constitutiveexpression of a complement-like protein in Toll and JAK gain-of-function mutants ofDrosophila. Proc. Natl. Acad. Sci. USA 97:11427-11432.

58. Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K., Natori, S., Kawabata, S., Iwanaga,S., Lee, B.L. 1998. Molecular cloning of cDNA for pro-phenol-oxidase-activating factor I, aserine protease is induced by lipopolysaccharide or 1,3-beta-glucan in coleoptera insect,Holotrichia diomphalia larvae. Eur. J. Biochem. 257:615-621.

59. Lee, S.Y., Wang, R., Söderhäll. K. 2000. A lipopolysaccharide- and β-1,3-glucan bindingprotein from hemocytes of the freshwater crayfish, Pacifastacus leniusculus. Purification,characterization and cDNA cloning. J. Biol. Chem. 275:1337-1343.


40

60. Lee, W.J., Lee, D.J., Kravchenko, V.V., Ulevitch., R.J., Brey, P.T. 1996. Purification andmolecular cloning of an inducible Gram negative bacteria-binding protein from the silkwormBombyx mori. Proc. Natl. Acad. Sci. USA 93:7888-7893.

61. Lee, S.Y., Söderhäll, K. 2001. Characterization of a pattern recognition protein, aMasquerade-like protein, in the freshwater crayfish Pacifastacus leniusculus. J. Immunol.166:7319-7326.

62. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J.M., Hoffmann, J.A. 1996. Thedorsoventral regulatory gene cassette spatzle/Toll/cactus control the potent antifungal response inDrosophila adults. Cell 86:973-983.

63. Levashina, E.A., Moita, L.F., Blandin, S., Vriend, G., Lagueux, M., Kafatos, F. 2001.Conserved role of a complement-like protein in phagocytosis by dsRNA knockout in culturedcells of the mosquito, Anopheles gambiae. Cell 104:709-718.

64. Liu, T., Lin, Y., Cislo, T., Minetti, C.A.S.A., Baba, J.M.K., Liu, T.Y. 1991. Limunectin. J.Biol. Chem. 266:14813-14821.

65. Lohman, J.U., Endl, I., Bosch, T.C.G. 1999. Silencing of the developmental genes in Hydra.Develop. Biol. 214:211-214.

66. Lubzens, E., Ravid, T., Khayat, M., Daube, N., Tietz, A. 1997. Isolation and characterizationof the high-density lipoproteins from the hemolymph and ovary of the penaeid shrimp, Penaeussemisulcatus (de Haan): apoproteins and lipids. J. Exp. Zool. 278:339-348.

67. Ma, C., Kanost, M. 2000. A β-1,3-glucan recognition from an insect, Manduca sexta,agglutinates microorganisms and activates the phenoloxidase cascade. J. Biol. Chem.275(11):7505-7514.

68. Manetti, R., Annunziato, F., Tomasevic, L., Gianno, V., Parronchi, P., Romagnani, S., Maggi,E. 1995. Polyinosinic acid: polycytidylic acid promotes T helper type 1-specific immuneresponses by stimulating macrophage production of interferon-alpha and interleukin-12. Eur. J.Immunol. 25:2656-2660.

69. Matsushita, M., Endo, Y., Nonaka, M., Fujita, T. 1998. Complement-related serine proteasesin tunicates and vertebrates. Curr. Opin. Immunol. 10(1):29-35.

70. Matsushita, M., Fujita, T. 1995. Cleavage of the third component of complement (C3) bymannose-binding protein-associated serine protease (MASP) with subsequent complementactivation. Immunobiol. 194:443-448.

71. Medzhitov, R., Janeway, C.A. 1997. Innate immunity: The virtues of a nonclonal system ofrecognition. Cell 91:295-298.

References

41

72. Medzhitov, R.M., Preston-Hurlburt, P., Janeway, C.A.Jr. 1997. A human homologue of theDrosophila Toll protein signals activation of adaptive immunity. Nature 388:394-397.

73. Meurs, E., Chong, K., Galabru, J., Thomas, N.S., Kerr, I.M., Williams, B.R., Hovanessian,A.G. 1990. Molecular cloning and characterization of the human double-stranded RNA-activatedprotein kinase induced by interferon. Cell 62:379-390.

74. Montgomery, M.K., Fire, A. 1998. Double-stranded RNA as a mediator in sequence-specificgenetic silencing and co-suppression. Trends Genet. 14:255-258.

75. Morishita, K., Tsuchiya, M., Asano, S., Kaziro, Y., Nagata, S. 1987. Chromosomal genestructure of human myeloperoxidase and regulation of its expression by granulocyte colony-stimulating factor. J. Biol. Chem. 262:15208-15213.

76. Muta, T., Hashimoto, R., Miyata, T., Nishimura, H., Toh, Y., Iwanaga, S. 1990. Proclottingenzyme from horseshoe crab hemocytes. CDNA cloning, disulfide locations, and subcellularlocalization. J. Biol. Chem. 265:22426-22433.

77. Muta, T., Miyata, T., Misumi, Y., Tokunaga, F., Nakamura, T., Toh, Y., Ikehara, Y.,Iwanaga, S. 1991. Limulus factor C: An endotoxin-sensitive serine protease zymogen with amosaic structure of complement-like, epidermal growth factor-like, and lectin-like domains. J.Biol. Chem. 266:6554-6561.

78. Muta, T., Oda, T., Iwanaga, S. 1993. Horseshoe crab coagulation factor B. A unique serineprotease zymogen activated by cleavage of an Ile-Ile bond. J. Biol. Chem. 268:21384-21388.

79. Müller, H.M., Dimopoulos, G., Blass, C., Kafatos, F.C. 1999. A hemocyte-like cell lineestablished from the malaria vector Anopheles gambiae express six prophenoloxidase genes. J.Biol. Chem. 274:11727-11735.

80. Müller, U., Steinhoff, U., Reis, L.F.L., Hemmi, S., Pavlovic, J., Zinkernagel, R.M., Aguet, M.1994. Functional role of type I and type II interferons in antiviral defense. Science 264:1918-1921.

81. Nagai, T., Kawabata, S.I. 2000. A link between blood coagulation and prophenoloxidaseactivation in arthropod host defense. J. Biol. Chem. 275:29264-29267.

82. Nappi, A., Vass, E. 1993. Melanogenesis and the generation of cytotoxic molecules duringinsect cellular immune reactions. Pigm. Cell Res. 6:117-126.

83. Natori, S., Kubo, T. 1996. Roles of lectin in development and morphogenesis in insects. In:New Directions in Invertebrate Immunology, Söderhäll, K., Iwanaga, S., Vasta, G.R. (Eds.), SOSPublications, Fair Haven. pp. 175-187.


42

84. Nelson, R.E., Fessler, L.I., Takagi, Y., Blumberg, B., Keene, D.R., Olson, P.F., Parker, C.G.,Fessler, J. 1994. Peroxidasin: a novel enzyme-matrix protein of Drosophila development. EMBOJ. 13:3438-3447.

85. Ochiai, M., Ashida, M. 1999. A pattern recognition protein for peptidoglycan: cloning thecDNA and the gene of the silkworm, Bombyx mori. J. Biol. Chem. 274:11854-11858.

86. Ochiai, M., Ashida, M. 2000. A pattern recognition protein for β-1,3-glucan. The bindingdomain and the cDNA cloning of β-1,3-glucan recognition protein from the silkworm, Bombyxmori. J. Biol. Chem. 275:4995-5002.

87. Parkinson, N., Smith, I., Weaver, R., Edwards, J.P. 2001. A new form of arthropodphenoloxidase is abundant in venom of the parasitoid wasp Pimpla hypochondriaca. InsectBiochem. Mol. Biol. 31:57-63.

88. Perrazolo, L.M., Barracco, M.A. 1997. The prophenoloxidase activating system of the shrimpPenaeus paulensis and associated factors. Dev. Comp. Immunol. 21:385-395.

89. Persson, M., Cerenius, L., Söderhäll, K. 1987. The influence of haemocyte number on theresistance of the freshwater crayfish, Pacifastacus leniusculus Dana, to the parasitic fungusAphanomyces astaci. J. Fish Dis. 10:471-477.

90. Pestka, S., Langer, J.A., Zoon, K.C., Samuel, C.E. 1987. Interferons and their actions. Annu.Rev. Biochem. 56:727-777.

91. Rantamäki, J., Durrant, H., Liang, Z., Ratcliffe, N.A., Duvic, B., Söderhäll, K. 1991. Isolationof a 90 kDa protein from haemocytes of Blaberus craniifer which has similar properties to the 76kDa protein from crayfish haemocytes. J. Insect Physiol. 37:627-634.

92. Rock, F.L., Hardiman, G., Timans, J.C., Kastelein, R.A., Bazan, J.F. 1998. A family ofhuman receptors structurally related to Drosophila Toll. Proc. Natl. Acad. Sci. USA 95:588-593.

93. Rosenberry, B. (ed.) 1995. Worlds shrimp farming 1995. Shrimp News International, SanDiego, CA, USA.

94. Rosenberry, B. (ed.) 1998. Worls shrimp farming 1998. Shrimp News International, SanDiego, CA, USA.

95. Rowley, A.F., Brookman, J.L., Ratcliffe, N.A. 1990. Possible involvement of theprophenoloxidase system of the locust, Locusta migratoria, in antimicrobial activity. J. Invertbr.Pathol. 56:31-38.

96. Ruoslahti, E. 1996. RGD and other recognition sequences for integrins. Annu. Rev. Dev. CellBiol. 12:697-715.

References

43

97. Sanchez-Alvarado, A., Newmark, P.A. 1999. Double-stranded RNA specifically disruptsgene expression during planaria regeneration. Proc. Natl. Acad. Sci. USA 96:5049-5054.

98. Sato, S., Masuya, H., Numakunai, T., Satoh, N., Ikeo, K., Gojobori, T., Tamura, K., Ide, H.,Takeuchi, T., Yamamoto, H. 1997. Ascidian tyrosinase gene: its unique structure and expressionin the developing brain. Dev. Dyn. 208:363-374.

99. Satoh, D., Horii, A., Ochiai, M., Ashida, M. 1999. Prophenoloxidase-activating enzyme ofthe silkworm, Bombyx mori: purification, characterization and cDNA cloning. J. Biol. Chem.274:7441-7453.

100. Seki, N., Muta, T., Oda, T., Kuma, K., Miata, T., Iwanaga, S. 1994. Horseshoe crab (1,3) β-D-glucan-sensitive coagulation factor G. A serine protease zymogen heterodimer with similaritiesto β-glucan-binding proteins. J. Biol. Chem. 269:1370-1374.

101. Schumann, R.R., Leong, S.R., Flaggs, G.W., Gray, P.W., Wright, S.D., Mathison, J.C.,Tobias, P.S., Ulevitch, R.J. 1990. Structure and function of lipopolysaccharide binding protein.Science 249:1429-1431.

102. Sharp, P.A. 1999. RNAi and double-stranded RNA. Genes Develop. 12:139-141.

103. Shin, S.W., Park, S.S., Park, D.S., Kim, M.G., Kim, S.C., Brey, P.T., Park, H.Y. 1998.Isolation and characterization of immune-related genes from the fall webworm, Hyphantriacunea, using PCR-based differential display and substractive cloning. Insect Biochem. Mol. Biol.28:827-837.

104. Sijen, T., Kooter, J.M. 2000. Post-transcriptional gene-silencing: RNAs on the attack or onthe defense. Bioessays 22:520-531.

105. Smith, L.C., Azumi, K., Nonaka, M. 1999. Complement systems in invertebrates. Theancient alternative and lectin pathways. Immunopharmacology 42:107-120.

106. Sritunyalucksana, K., Cerenius, L., Söderhäll, K. 1999. Molecular cloning andcharacterization of prophenoloxidase in the black tiger shrimp, Penaeus monodon. Dev. Comp.Immunol. 23:179-186.

107. Sritunyalucksana, K., Lee, S.Y., Söderhäll, K. 2001. A β-1,3-glucan binding protein fromthe black tiger shrimp, Penaeus monodon. Dev. Comp. Immunol. (in press).

108. Sritunyalucksana, K., Söderhäll, K. 2000. The proPO and clotting system in crustaceans.Aquaculture 191:53-69.


44

109. Sritunyalucksana, K., Wongsuebsantati, K., Johansson, M.W., Söderhäll, K. 2001.Peroxinectin, a cell adhesive protein associated with the proPO system from the black tigershrimp, Penaeus monodon. Dev. Comp. Immunol. 25:353-363.

110. St. Leger, M., Cooper, R.M., Charnely, A.K. 1988. The effect of melanization of Manducasexta cuticle on growth and infection by Metarhizium anisopliae. J. Invertebr. Pathol. 52:459-470.

111. Sugumaran, M. 1996. Roles of the insect cuticle in host defense reactions. In: NewDirections in Invertebrate Immunology, Söderhäll, K., Iwanaga, S., Vasta, G.R. (Eds.), SOSPublications, Fair Haven. pp. 355-374.

112. Sun, S.C., Lundström, I., Boman,H.G., Faye, I., Schmidt, O. 1990. Hemolin: an insectimmune protein belonging to the immunoglobulin superfamily. Science 250:1729-1732.

113. Söderhäll, K. 1982. Phenoloxidase activating system and melanization- a recognitionmechanism of arthropods? A review. Dev. Comp. Immunol. 6:601-611.

114. Söderhäll, K., Ajaxon, R. 1982. Effect of quinone and melanin on mycelial growth ofAphanomyces spp. and extracellular protease of Aphanomyces astaci, a parasite of crayfish. J.Invertbr. Pathol. 39:105-109.

115. Söderhäll, K., Cerenius, L. 1998. Role of the prophenoloxidase activating system ininvertebrate immunity. Curr. Opin. Immunol. 10:23-28.

116. Söderhäll, K., Cerenius, L., Johansson, M.W. 1994. The prophenoloxidase activating systemand its role in invertebrate defense. Ann. N. Y. Acad. Sci. 712:155-161.

117. Söderhall, I., Söderhäll, K. 2001. A method for long-time culture of hematopoietic cellsfrom the freshwater crayfish and penaeid shrimp. Patent application.

118. Tabara, H., Grishok, A., Mello, C.C. 1998. RNAi in C. elegans: Soaking in the genomesequence. Science 282:430-431.

119. Thörnqvist, P.O., Johansson, M.W., Söderhäll, K. 1994. Opsonic activity of cell adhesionproteins and β-1,3-glucan-binding proteins from two crustaceans. Dev. Comp. Immunol. 18:3-12.

120. van der Broek, M.F., Muller, U., Huang, S., Aguet, M., Zinkernagel, R.M. 1995. Antiviraldefense in mice lacking alpha-beta and gamma interferon receptors. J. Virol. 69:4792-4796.

121. Vargas-Albores, F., Guzman, M.A., Ochoa, J.L. 1993. A lipopolysaccharide bindingagglutinin isolated from brown shrimp (Penaeus californiensis Holmes) haemolymph. Comp.Biochem. Physiol. 104B:407-413.

References

45

122. Vargas-Albores, F., Jimenez-Vega, F., Söderhäll, K. 1996. A plasma protein isolated frombrown shrimp (Penaeus californiensis) which enhances the activation of prophenoloxidasesystem by β-1,3-glucan. Dev. Comp. Immunol. 20:299-306.

123. Vargas-Albores, F., Jiménez-Vega, F., Yepiz-Placencia, G.M. 1997. Purification andcomparison of β-1,3-glucan binding protein from white shrimp (Penaeus vannamei). Comp.Biochem. Physiol. 116:5453-458.

124. Verdijk, R.M., Mutis, T., Esendam, B., Kamp, J., Melief, C.J., Brand, A., Goulmz, E. 1999.Polyriboinosinic polyribocytidylic acid (poly (I:C)) induces stable maturation of functionallyactive human dendritic cells. J. Immunol. 163:57-61.

125. Volanakis, J.E. 1998. Overview of the complement system. In: The human complementsystem in health and disease, Volanakis, J.E. and Frank, M.M. (Eds.), New York: Marcel Dekker,Inc. pp. 9-32.

126. Wang, R., Lee, S.Y., Cerenius, L., Söderhäll, K. 2001. Properties of the prophenoloxidaseactivating enzyme of the freshwater crayfish, Pacifastacus leniusculus. Eur. J. Biochem.268:895-902.

127. Wianny, F., Zernicka-Goetz, M. 2000. Specific interference with gene function by double-stranded RNA in early mouse development. Nat. Cell Biol. 2:70-75.

128. Yeh, M.S., Huang, C.J., Leu, J.H., Lee, Y.C., Tsai, I.H. 1999. Molecular cloning andcharacterization of a hemolymph clottable protein from tiger shrimp (Penaeus monodon). Eur. J.Biochem. 266:624-633.

129. Yepiz-Plascencia, G.M., Vargas-Albores, F., Jimenez-Vega, F., Ruiz-Verdugo, L.M.,Romo-Figueroa, G. 1998. Shrimp plasma HDL and β-glucan binding protein (BGBP):comparison of biochemical characteristics. Comp. Biochem. Physiol. 121B:309-314.

130. Yoshida, H., Kinoshita, K., Ashida, M. 1996. Purification of a peptidoglycan recognitionprotein from the hemolymph of the silkworm, Bombyx mori. J. Biol. Chem. 271:13854-13860.

131. Zeng, J., Fenna, R.E. 1992. X-ray crystal structure of canine myeloperoxidase at 3 A°resolution. J. Mol. Biol. 226:185-207.

Characterisation of Some Immune Genes in the Black Tiger - DiVA

Documents

Transcript of Characterisation of Some Immune Genes in the Black Tiger - DiVA