Fish & Shellﬁsh Immunology - GBC - Global Biotech ...€¦ · aCenter of Excellence for Molecular...

at SciVerse ScienceDirect

Fish & Shellfish Immunology xxx (2012) 1e14

Contents lists available

Fish & Shellfish Immunology

journal homepage: www.elsevier .com/locate / fs i

Discovery of immune molecules and their crucial functions in shrimp immunity

Anchalee Tassanakajon a,*, Kunlaya Somboonwiwat a, Premruethai Supungul a,b, Sureerat Tang a,b

aCenter of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road,Bangkok 10330, ThailandbNational Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand

a r t i c l e i n f o

Article history:Received 24 July 2012Received in revised form21 September 2012Accepted 24 September 2012Available online xxx

Keywords:Penaeid shrimpInnate immunityImmune molecules

* Corresponding author. Tel.: þ66 2 218 5439; fax:E-mail address: [email protected] (A. Tassana

1050-4648/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.fsi.2012.09.021

Please cite this article in press as: TassanakajShellfish Immunology (2012), http://dx.doi.o

a b s t r a c t

Several immune-related molecules in penaeid shrimps have been discovered, most of these via theanalysis of expressed sequence tag libraries, microarray studies and proteomic approaches. Theseimmune molecules include antimicrobial peptides, serine proteinases and inhibitors, phenoloxidases,oxidative enzymes, clottable protein, pattern recognition proteins, lectins, Toll receptors, and otherhumoral factors that might participate in the innate immune system of shrimps. These molecules havemainly been found in the hemolymph and hemocytes, which are the main sites where immune reactionstake place, while some are found in other immune organs/tissues, such as the lymphoid organs, gills andintestines. Although the participation of some of these immune molecules in the shrimp innate immunedefense against invading pathogens has been demonstrated, the functions of many molecules remainunclear. This review summarizes the current status of our knowledge concerning the discovery andfunctional characterization of the immune molecules in penaeid shrimps.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Penaeid shrimps include some economically important andaquacultured marine species, such as the Pacific white shrimpLitopenaeus vannamei and the black tiger shrimp Penaeus monodonthat are currently the two main successfully species culturedworldwide. However, disease outbreaks have caused massivemortality and a great loss to the shrimp cultivation industry, andparticularly from the outbreaks caused by the major shrimp path-ogens of white spot syndrome virus (WSSV), yellow head virus(YHV), infectious myonecrosis virus (IMNV), and bacteria in thegenus Vibrio [1,2]. Understanding the innate immune responses ofshrimps against invading microbes provides essential informationfor the establishment of effective methods to control these and,potentially, those of related emerging infectious diseases.

Lacking an adaptive immune system, shrimps rely on theireffective cellular and humoral innate immune responses to combatinvading microbes [3]. The cellular immune reactions includephagocytosis, nodulation and encapsulation, whereas the humoralresponses involve the synthesis and release of several immuneproteins, such as antimicrobial peptides (AMPs), proteinase inhib-itors, cytokine-like factors, etc. In crustaceans, including shrimps,

þ66 2 218 5418.kajon).

All rights reserved.

on A, et al., Discovery of immrg/10.1016/j.fsi.2012.09.021

major immune reactions take place in hemolymph, which containsthree different principal types of hemocytes that are defined as thehyaline, granular and semigranular hemocytes [4]. Several immunemolecules are produced and stored in the granules of hemocytesbefore being released into the hemolymph upon activation bybacterial and/or fungal cell wall components, such as peptidoglycan(PG), lipopolysaccharides (LPS) and b-glucans (BGs) [5]. Patternrecognition proteins (PRPs) or pattern recognition receptors (PRRs)recognize and bind the microbial cell wall components and activatevarious immune responses [6e8].

In this review, we describe the discovery of immune-relatedmolecules by the high throughput technologies of genomic andproteomic analyses and the characterization of these immunemolecules that participate in the major immune reactions againstinvading pathogens in shrimp.

2. Discovery of immune genes/proteins by high throughputgenomic/proteomic approaches

2.1. Expressed sequence tag (EST) analysis of immune genes inpenaeid shrimp

Shrimp have a relatively large genome size of approximately2 � 109 bp [9], which in part reflects the high percentage ofrepetitive sequences [10]. The whole genome sequencing ofL. vannamei is now in progress but the information is not yet

une molecules and their crucial functions in shrimp immunity, Fish &

Delta:1_given name

Delta:1_given name

Delta:1_surname

Delta:1_given name

Delta:1_given name

Delta:1_surname

Delta:1_given name

Delta:1_given name

mailto:[email protected]

mailto:imprint_logo

www.sciencedirect.com/science/journal/10504648

http://www.elsevier.com/locate/fsi

mailto:journal_logo

http://dx.doi.org/10.1016/j.fsi.2012.09.021



Table 1Immune-related genes of penaeid shrimps initially identified by expressed sequencetag (EST), suppression subtractive hybridization (SSH) and microarray as well astheir characterized functions.

Immune-relatedgenes

Tissuedistributiona

Stressresponseb

Function

1. Antimicrobial peptidesAntilipopolysaccharide

factorsG, Hc, Lo dsRNA, H,

N, V, W, YAntimicrobial activity,antiviral activity,antifungal activity

Bactinecin Hc W NDCrustins Ep, G, Hc AA, H, N,

P, W, V, YAntimicrobialactivity

Lysozymes G, Hc, Hp,Ht, Lo

dsRNA, N,W, V, H, P

Antimicrobial activity

Penaeidins Hc AA, H, N,V, W, Y

Antimicrobial activity,antifungal activity

Single whey acidicproteindomain-containingpeptides

Hc N, P, W Antimicrobial activity

2. ProPO systemMasquerade-like serine

proteinase-like proteinHc W, Y, V, P Mediates hemocyte

adhesion, binding tobacterial cell wall,antimicrobial activity

Prophenoloxidase Hc AA, H, N, P,V, W, Y

Clearing the bacteriafrom circulation afterinfection

Prophenoloxidaseactivating factor

Hc, Lo H, N, V, W, Y Clearing the bacteriafrom circulation afterinfection

3. Oxidative stressCopper chaperone Hc N NDCopper/zinc superoxide

dismutaseHc W, Y ND

Cytosolic MnSOD Hc, Hp H, N, V, W NDDeath associated protein

diphenol oxidaseHc N ND

Glutathione peroxidase Hc H NDGlutathione-S-transferase G, Hc, Hp N, V, W, Y NDHeme peroxidase Lo N, V, W NDPeroxiredoxin Hp N Antioxidant activityPeroxisomal antioxidant

enzymeHc N ND

Thioredoxin C, G, Hp N, W Antioxidant activityThioredoxin reductase G, Hp W ND4. Proteinases/proteinase inhibitorsAlpha-2-macroglobulin Hc AA, P, W, Y Inhibit fibrinolytic

activity of bacteriaAminopeptidase G, Hc N, W NDAntileukoprotease Hc W NDAstacin protease Hp W NDCaspase Hp W Protease,

WSSV-inducedapoptosis

Cathepsin A Hc, Lo N, V, W NDCathepsin C Lo N, V NDCathepsin D Hp, Lo N, W Muscle proteasesCathepsin L G, Hc,

Hp, Lo,AA, dsRNA,N, V, W, Y

ND

Cathepsis B Hc, Lo N, V ProteaseChelonianin Hc W Antimicrobial activityCubilin protease G W NDCystein protease

caspase-2Lo N, V, W ND

Double whey acidicdomain-containingpeptide

Hc Proteinase inhibitor,antimicrobial activity

Elastase inhibitor Hc W NDGene MAC25 protein Hc H, N, V NDKunitz-type inhibitor Hc W Inhibit the activity

of trypsinLeucocyte elastase

inhibitorG, Hc,Hp, Lo

V ND

Lysosomalcaboxypeptidase

Hc W ND

A. Tassanakajon et al. / Fish & Shellfish Immunology xxx (2012) 1e142

publically available. Rather, to date the information on the shrimpgenome has largely been obtained from the analysis of expressedsequence tags (ESTs) sequences. This has led to several tissue-specific transcripts being identified as well as candidate genesthat may be implicated in the shrimp immune responses (Table 1).Nevertheless, the function of these immune genes and proteins arepoorly understood and almost all require further studies to unveiltheir function in the shrimp immune system.

The analysis of EST libraries that has been generated fromvarious cells or tissues provides information on the tissue-specificprofiles of gene expression and relative transcript abundance that islikely to reflect the function of those cells or tissues. Likewise, thecomparison of EST libraries between different pathogen infected orcontrol tissues at various times after infection can provide infor-mation on genes whose transcript expression levels are signifi-cantly changed over time as a result of the infection.

The immune system of invertebrates has been well studied ininsects, including the outstanding model of the fruit fly Drosophilamelanogaster, for which a large number of deposited EST sequences(821,005 ESTs as of May, 2012) are available in public databases andbacked up by extensive gene characterization studies. Moreover,several immune-related genes from other insects have now beenidentified and characterized. In contrast, much less information iscurrently available for crustaceans including shrimps. For penaeidshrimps, there are only 216,436 EST (0.3% of the total number ofESTs) sequences deposited in GenBank, and are comprised of161,241 ESTs from L. vannamei, 39,397 ESTs from P. monodon, 10,446ESTs from Fenneropenaeus chinensis and 5352 ESTs from otherpenaeid shrimps. To gain more information on the genomics ofshrimps, several ESTs have been constructed fromvarious tissues ofshrimps that were reared under normal, stressed or pathogen-challenged conditions, depending on their purposes [11e13]. In2011, Leu et al. [14] have constructed a shrimp transcriptomedatabase based on EST libraries of four major penaeid shrimpsincluding L. vannamei, P. monodon, F. chinensis and Marsupenaeusjaponicus. To isolate the shrimp immune-related genes, the large-scale EST libraries were established from three shrimp species,L. vannamei, P. monodon and F. chinensis [11e13]. O’Leary et al. [12],generated cDNA libraries derived from multiple tissues ofL. vannamei and these derived cDNA libraries depleted of theredundant transcripts. From the total of 13,656 ESTclones, 7896 ESTclones were randomly sequenced from six non-normalized cDNAlibraries that were derived from the hemocyte, hepatopancreas,gill, lymphoid organ, eyestalk and ventral nerve cord, respectively.A further 5760 EST clones were randomly selected from 34 differentsuppression subtractive hybridization (SSH) derived cDNA librariesgenerated from the hemocyte, gill and hepatopancreas, respec-tively, as they were predicted to be immune-related organs [15].Analysis of these sequences identified 7466 unique sequencesrepresented by 1981 contigs and 5485 singletons, and 38% of theunique genes were homologs of genes deposited in the GenBankdatabase. Nearly 40% of the EST clones were derived from hemo-cytes, which are likely to be the primary immune cells of shrimps.From these analyses, several potential immune genes were iden-tified, including AMPs, proteinase inhibitors, clottable protein andheat shock proteins (HSPs). Tassanakajon et al. [11] reported the ESTanalysis of 15 cDNA libraries from P. monodon, comprised of 13standard libraries and two normalized libraries. For the standardlibraries, six different tissues (hemocyte, hepatopancreas,lymphoid organ, eyestalk, hematopoietic tissue and ovary) fromhealthy and microbial-challenged or heat-stressed shrimps wereused to potentially identify candidate genes involved in theimmune defense, growth and sex differentiation. Additionally, twonormalized cDNA libraries were generated from the hepatopan-creas and lymphoid organ. The total of 10,100 EST clones were

Please cite this article in press as: Tassanakajon A, et al., Discovery of immune molecules and their crucial functions in shrimp immunity, Fish &Shellfish Immunology (2012), http://dx.doi.org/10.1016/j.fsi.2012.09.021

Table 1 (continued)

Immune-relatedgenes

Tissuedistributiona

Stressresponseb

Function

Neprilysinmethalloproteinase

G dsRNA ND

Papain familycystein protease

Hc W, Y Cleavage patternreminiscent ofbacterial collagenase

Prohormone andneuropeptideprocession protease

Hc N ND

Protease m1 zincmethalloprotease

Hc V, W ND

Proteinaseinhibitor-Kazaltype

C, Hc AA, H, N,P, V, W, Y

Proteinase inhibitor,antiviral activity

Serinecarboxypeptidase

Hp W ND

Serine protease Hc, Hp, Lo AA, H, N,V, W

Antimicrobial activity

Serine proteaseinhibitor

Hc dsRNA,N, W

Protease inhibitor

Serpin Hc N NDWAP domain protease

inhibitorHc dsRNA, N ND

Zinc proteinase Mpc1 Wh N, W NDZn carboxypeptidase G dsRNA, W ND5. Apoptotic tumor-related proteinAutophagy protein 9 Hp W NDCellular apoptosis

susceptibility proteinHp W ND

Defender against celldeath 1(DAD1)

Hc, Lo H ND

GULF adaptor protein Hp W NDIAP associated or Viaf1 Hc N NDPhosphatidylserine

receptor,phagocytosis ofapoptotic cells

Hc N ND

Proeasome 26Ssubunit

Hc W ND

Program cell deathprotein

Hc H, P, W ND

Rat insulinomagene-Rig

Hc P, W Apoptosis andtumor-related protein

RING-box protein2 (Rbx2)

Hc P Apoptosis andtumor-related protein

Survivin Hc, Lo H, N, V, W NDTranslational control

tumor proteinHc, Lo H, N, V, W ND

Tumor necrosis factor Lo N, V NDWilm’s tumor-related

protein-QMHc W ND

6. PRPPb-1,3-D-glucan-binding

proteinHp N, W Pattern recognition

protein of LPS andb-1,3-glucan in theshrimpproPO-activatingsystem, enhance thephenoloxidase activity

C-type lectin C, E, Hc,Hp, Wh

AA, N, V,W, Y

Agglutination

PRP chitin-bindinglectin

Hc V, W, Y ND

Perlucin Hc, Hp N NDPmAV Hc, Lo, Wh N, V, W Antiviral activityTachylectin Hc, Lo N, V, W ND7. Blood clotting systemClottable protein G, Hc, Hp AA, N, P,

WND

Coagulation factorVIII-associatedprotein

Hc Y ND

Coagulation factor XI Hc N ND

Table 1 (continued)

Immune-relatedgenes

Tissuedistributiona

Stressresponseb

Function

Proclotting enzymeprecursor

Hc H ND

Transglutaminase Hc, Lo H, N, P,W, V, Y

ND

8. Signaling transductionActivator of MAPK

pathwayHc N ND

cAMP dependentprotein kinase

G W ND

Casein kinase II, alphasubunit

Hc W ND

I-kappa-B kinase Hc N, W NDIMD Hc, Lo N, W NDInositol 1,4,5-triphosphate

3-kinaseHp I ND

Integrin alpha Hc dsRNA NDIntegrin beta Hc dsRNA NDInterleukin-1 receptor Hc H NDPeroxinectin G, Hc N, W NDPlatelet derived growth

factor-likeG, Hp W ND

Protein kinase C Hc N NDProtein kinase C

binding proteinHc N ND

Pulmonarysurfactant-associatedprotein D

Hc V ND

Putative regulator ofMAPKpathway

Hc, Hp N, W ND

Ras-like GTP-bindingprotein RHO

Hc H ND

Serine/threoninekinase

G, Hc, Hp dsRNA,N, W

ND

Serine/threonineproteinphosphatase

G, Hc, Hp N, W ND

Src-family tyrosineprotein kinase

Hp W ND

STAT Hc N NDTyrosine kinase G N NDWD40-domain G, Hc, Hp N NDZn finger protein

associatedwith PKC-relatedkinase

Hp W ND

9. Heat shock proteinsChaperonin Lo N, V NDChaperonin containing

T-complexHc, Lo H, N,

V, WND

Co-chaperone proteinHscB

Hc, G W ND

Copper homeostaosisprotein

Lo N ND

Cyclophilin 18 Lo N, V NDCyclophilin 5 Lo N, V NDCyclophilin A Lo N, V NDDnaJ domain Hc N NDDnaK protein Hc W NDHSP cognate 28 Hc N NDHSP cognate 29 Hc N NDHSP cognate 3 Hp N NDHSP cognate 5 Hc, Hp N, W NDHSP cognate 70 C N, W NDHSP10 Hc, Hp N NDHSP60 C, G N, W NDHSP70 E, G, Hc, Lo H, N,

V, W, YND

HSP82 Hc N NDHSP90 C, E, Hc N, W, Y NDHSPA and B Hp N ND

(continued on next page)

A. Tassanakajon et al. / Fish & Shellfish Immunology xxx (2012) 1e14 3

Please cite this article in press as: Tassanakajon A, et al., Discovery of immune molecules and their crucial functions in shrimp immunity, Fish &Shellfish Immunology (2012), http://dx.doi.org/10.1016/j.fsi.2012.09.021

Table 1 (continued)

Immune-relatedgenes

Tissuedistributiona

Stressresponseb

Function

Peptidyleprolylcisetrans-isomerase

Hc H ND

10. Other immune moleculesCadherin Hp W NDCalcium-regulated

heat-stableprotein

Hc H, W ND

Calreticulin precursor C, G, Hc, Hp N, W NDCCCH-type zinc finger

antiviralprotein

Hc, Hp V, W, Y Antiviral activity

Chitin-bindingperitrophin-A

Wh W ND

Chitinase Hp N, W NDComplement

component 1Hc H ND

Crustacyanin Lo N NDCu-metallothionin Hp N NDCyclophilin Hc H, N, V NDCyclophilin

peptidyl-prolylHc, Hp N ND

Fc fragment of IgG Hc H, V NDFerritin G, Hc,

Hp, WhAA, H, N,V, W, Y

ND

Ficolin 1-like Hc V, W, Y Agglutinationg-interferon inducible

lysosomalthiol reductase-likeprotein

Hc W, Y ND

Hemocyanin C, Hp, Wh AA, N,W

Oxygen transfer,antimicrobial activity

Histone H1-beta Hc W, Y Packaging ofgenomic DNA,antimicrobial activity

Histone H2A variant Z Hc W Packaging ofgenomic DNA,antimicrobialactivity

Histone H3, family Hc N Packaging ofgenomic DNA

Histone H4 Hc W Packaging ofgenomic DNA,antimicrobialactivity

HLA-B associatedtranscript-3isoform a

Hc H ND

IK cytokine Lo N NDImmunoglobulin

domainHc W ND

ImmunophilinFKBP-52

Hc H ND

KIN 17 protein Hc H NDMacrophage

mannosereceptor

Hp W ND

Macrophagemigrationinhibitory factor

Lo N ND

Metallothionein 1 C, Hc, Hp N, Y NDMinus agglutinin Hc V, W, Y NDPeritrophin C, G,

Hc, LoN, V, W Antimicrobial activity

PKR-associatingprotein RAX

E N ND

Platelet derivedgrowthfactor-like

G, Hp W ND

Presenilin enhancer Hc H NDProfilin Lo AA, N, V NDSelenoprotein H G W NDSelenoprotein M G W AntioxidantSelenoprotein W1 Hc, Hp N, Y NDTetraspanin G, Hp N, W ND

Table 1 (continued)

Immune-relatedgenes

Tissuedistributiona

Stressresponseb

Function

Thrombospondin C, G, Hc,Hp, Lo

N, V, W,Y

ND

Thymosin Hc, Lo AA, H, V,W, Y

ND

Tick legumain Hc W, Y NDTrehalose-phosphate

synthaseC N, W ND

Vesiclemannose-bindinglectin

Hc W ND

a C ¼ cephalothorax, E ¼ eyestalk, Ep ¼ epipodite, G ¼ gill, Ht ¼ heart,Hc ¼ hemocyte, Hp ¼ hepatopancreas, Lo ¼ lymphoid organ, Wh ¼ whole shrimp.

b Data obtained from EST and microarray analyses. AA ¼ antibiotic administra-tion, P¼ peptidoglycan stimulation, N¼ normal, W¼WSSV, Y¼ YHV, V¼ Vibrio sp.,H ¼ heat stress.


Please cite this article in press as: Tassanakajon A, et al., Discovery of immShellfish Immunology (2012), http://dx.doi.org/10.1016/j.fsi.2012.09.021

obtained and clustered into 917 contigs and 3928 singletons,represent 4845 unique sequences (available at http://pmonodon.biotec.or.th). Of these, 49% matched were homologous to existingsequences at GenBank. Putative immune genes were predominantin all the hemocyte libraries (10e13%), but showed differentexpression profiling between the normal condition and the statesof different pathogen infections or heat stress treatment.

Leu et al. [16] reported the EST analysis of normal and WSSV-infected cDNA libraries from P. monodon postlarvae. A total of6964 and 7686 EST clones from normal and viral-infected librarieswere sequenced, assembled and clustered into 9622 uniquesequences composed of 1364 contigs and 8258 singletons. Afterhomology searches, 45% (3022) of the ESTs from the normal librarymatched to the deposited genes in the GenBank database and 43%(2870) matched to protein sequences in the UniProt database. Inthe viral-infected library, the matched ESTs revealed 46% (3338ESTs) and 44% (3202 ESTs) homologs with the genes deposited inboth databases. In F. chinensis, the large-scale collection of ESTs wasgenerated from a cephalothorax cDNA library [12]. A total of 10,446ESTs were randomly sequenced and, after assembling and clus-tering, contained 1399 contigs and 1721 singletons, yielding 3120unique sequences. Of these unique sequences, 1373 (44%) werematched to those in GenBank, and 81 were likely to be immune-related genes.

Shrimp DNA microarrays containing unique sequences havebeen constructed based on the information of the EST sequencesand used to investigate the gene expression profile after pathogen-challenge [15,17e23], treatment with antibiotic or PG [24,25], andenvironmental stress [26]. In addition, the gene expression profilein thematuring ovary in response to eyestalk ablationwas analyzedby microarray in P. monodon [27]. Microarray-based analyses ofshrimp immune responses have recently reviewed by Aoki et al.[17]. The analysis of shrimp immune genes that responded uponWSSV infection [18] revealed genes in the cephalothorax ofF. chinensis that were up-regulated after WSSV infection. A cDNAmicroarray of P. monodon was also constructed to study the geneexpression levels of hemocytes upon WSSV infection [21], whilstthe transcriptomes of the hepatopancreas, hemocyte, muscle andgill tissues after WSSV challenge have been investigated [15].

Microarray analyses revealed the differential gene expressionprofiles in shrimp tissues depended on the type of tissues andinfectious pathogens. Wang et al. [19] compared the gene expres-sion profile of F. chinensis challenged with WSSV and those chal-lenged with heat-inactivated Vibrio anguillarum, whilstPongsomboon et al. [22] analyzed the expression of gene tran-scripts upon viral (WSSV and YHV) and bacteria (Vibrio harveyi)


http://pmonodon.biotec.or.th

http://pmonodon.biotec.or.th


infections. The total number of differentially expressed genes afterWSSV or YHV infection was 4.7- and 2.07-fold higher, respectively,than that of V. harveyi-challenged P. monodon, and 2.5-fold higherin WSSV-infected F. chinensis compared to that of heat-inactivatedV. anguillarum [19,22]. These revealed that viral infections mayaffect the transcript levels of a greater number of host genes thanthe bacteria do. Moreover, the numbers of differentially expressedgenes were higher in WSSV-challenged shrimp than in the YHV-challenged ones. Many immune molecules, including anti-lipopolysaccharide factor (ALF) and lysozyme, were significantlyup-regulated at 6 h after V. harveyi-challenge [28], whilst a numberof the differentially expressed genes in bacterial infection werehighest at the early stages of infection. This also illustrates that theinnate immune system responds rapidly to pathogen invasion atthe level of altered gene expression, the effects being noticedwithin the first hour to days after infection [3]. In contrast, thenumber of altered expression genes, including ferritin, ficolin,PmAV, proteinase inhibitor, carboxylesterase-6, arginase, tran-scriptional or translational proteins and ribosomal proteins, by viralmodulators were highest at the late infection stage (48 h post-infection (hpi)). The up-regulation of these genes suggests that theymay be involved in shrimp antiviral responses at the late stage ofinfection or tissue repair following viral- or immune response-induced cell damage or death. In addition, shrimps at the moribundstage might then express genes that are induced or hijacked by thevirus so as to use the host cell machinery for their replication andproduction of new virions.

Interestingly, several unknown genes have been found to behighly altered in expression levels in response to stress/pathogeninduction. Clearly, the function of these unknown gene productsneeds to be further investigated. Although EST and microarrayanalysis are limited, as, for example, important immune mediatorsthat do not alter significantly in transcript expression levels but arepost-translationally modified or are involved in the early parts ofsignal cascades and at low expression levels, these approachescover most of the current knowledge of shrimp immunity while thewhole genome analysis remains unavailable. However, to compli-ment these approaches, proteomic approaches are being employedas well as, but both are restricted by the absence of good trans-formation vectors and permanent cell lines, in contrast to insects,confining the subsequent analysis to the use of transient geneknockdown and heterologous gene expression studies and the useof yeast two-hybrid systems (see next section).

2.2. Proteomic analysis for the discovery of immune proteins

The study of shrimp immune responses is not only limited bya lack of genomic data, but also of a clear understanding of post-translational control. The identification, characterization andfunction ascribing of the significance and biological functions ofshrimp proteins have mainly been explored by genomic- andhomology-based technologies. However, the proteomics-basedapproach is more and more important to uncover novel proteinsand to characterize the actual function of shrimp proteins. Theabove section described the use of genomic approaches for geneidentification and characterization, and so here the proteomictechniques that have recently been applied for the study of shrimpproteins are described.

Proteomic techniques, such as two-dimensional gel electro-phoresis (2DGE) or two-dimensional liquid chromatographycoupled with mass spectrometry have been used to study theshrimp proteome and to monitor changes in protein synthesisduring environmental stresses and systemic infection by variouspathogenic microorganisms. Shotgun proteomics identified 11novel viral proteins and 429 host proteins from P. monodon


subcuticular epithelial cells infected withWSSV [29]. The lymphoidorgan proteome of healthy F. chinensis was analyzed by 2DGE andcould be classified into 13 categories, with themajority being foundto be cytoskeleton proteins [30].

The changes in protein expression during environmentalstresses, such as during hypoxic stress and crowding in intensiverearing ponds [31,32], were demonstrated. According to Silvestreet al. [32], the potential for the abundant hemocyanin and sarco-plasmic calcium-binding proteins in the hemolymph to be used asa biomarker for environmental stress levels in farmed P. monodonshrimps was suggested [32]. However, further studies are requiredto confirm and quantify this possibility.

Nevertheless, the main interest has been centered upon hostepathogen interactions, where knowledge of the responses at thetranslational level of pathogen-challenged shrimps could be usefulfor understanding further aspects of shrimp immunity and arecomplimentary to the genomic approaches. Comparative proteo-mic analyses have been employed to identify altered proteinexpression levels after viral and bacterial infections. For example,differentially expressed shrimp proteins uponWSSV infectionwereidentified from the subcuticular epithelium, gill, hepatopancreasand stomach tissues. Moreover, the proteins with altered expres-sion levels following WSSV infection in the epithelial lysate wereidentified using cleavable isotope-coded affinity tags [29].

Likewise, comparative analyses of the protein expressionpattern of the hepatopancreas and stomach in unchallenged andWSSV-challenged shrimp identified several differentiallyexpressed proteins [33,34]. Besides the host proteins that havebeen identified, a WSSV encoded protein (i.e. ICP11) was alsofound to be highly expressed in the stomach proteome, and thiscorresponded to its transcription level expression [34]. Furtherstudy suggested that ICP11 acts as a DNAmimic that prevents DNAfrom binding to histone proteins H2A, H2B, H3 and H2A.x in thehemocytes of WSSV-infected shrimp and, thus, disrupts nucleo-some assembly. ICP11 also colocalized with histone H3 andactivated-H2A.x, suggesting that ICP11 might cause impairment ofDNA repair following double strand breaks [35]. In a similarmanner, significant alteration of protein expression levels in thelymphoid organ and gills of P. monodon upon YHV infection and inthe hemocytes of L. vannamei upon Taura syndrome virus (TSV)infection was also reported [36e38].

Apart from the major viral pathogens, bacteria in the Vibriogenus are another severe pathogen and are responsible for causingvibriosis. Analysis of the differentially expressed proteins in themajor immune tissues, such as the hemocyte and lymphoid organ,of V. anguillarum-challenged F. chinensis and V. harveyi-challengedP. monodon shrimp revealed clear differences [30,39,40]. Forexample, some were up-regulated but the majority was down-regulated upon Vibrio challenge and a high proportionwas found tobe cytoskeletal proteins. Vibrio infection also triggered changes inthe expression levels of more traditionally recognized immune-related proteins, from which phagocytosis and the prophenolox-idase (proPO) activating system were reported to be the principalimmune components with a crucial role in response to bacterialchallenge.

From the proteomic analysis, typical scenarios of how shrimpmight respond to pathogens and environmental stresses wereinferred. To gain more insight into the functions of each specificprotein, other proteomic methods, such as the yeast two-hybridassay and in vitro pull-down assay have been used to isolateinteracting partners, which could lead to functional characteriza-tion of the proteins in shrimp. For example, the yeast two-hybridassay revealed that the shrimp laminin receptor specifically inter-acted with capsid/envelope proteins of TSV, IMNV and YHV, and sois a potential receptor for these viruses in shrimp [41,42]. Pm-



fortilin is an anti-apoptotic protein identified in P. monodon thatexhibits antiviral properties. Screening for the cellular proteinpartners that it interacts with revealed a putative antiviral peptidecontaining XPPX signature sequences [43]. However, even whenthe interacting proteins have been potentially identified, thefunction of the proteins may still remain mysterious. For instance,the apoptosis-related gene, PmAlix, identified from P. monodon, wasfound to bind to a predicted ubiquitin C and to a putative guanylylcyclase. Nevertheless, the exact role of PmAlix and its interactingpartners has yet to be elucidated [44].

In different circumstances, to study the hostepathogen inter-actions, viral proteins have been used as a bait protein to screen forthe interacting host protein. For example, the major nucleocapsidprotein of WSSV (VP15) that is involved in packaging of the WSSVgenome during virion formation was used to search for P. monodonproteins that are functionally related. A protein named PmFKBP46was found to colocalize with VP15 in the nucleus and might beinvolved in the genome packaging of the virus during virionassembly [45], although this still awaits confirmation.

Thus, both genomic and proteomic approaches have identifiedseveral immune-relatedmolecules that respond, in terms of alteredexpression levels, to various stresses, including pathogen infec-tions. The major immune molecules and the defense reactions arenow described below.

3. The major immune molecules and the defense reactions

3.1. Antimicrobial peptides (AMPs)

AMPs are effectors of the innate immune system and functionas a first line of defense to fight against invading microorganisms[46]. Therefore, AMPs are critical for shrimp to fight against thepathogenic invasion. AMPs are typically small in size, generally lessthan 150e200 amino acid residues, and have an amphipathicstructure with cationic or anionic properties. AMPs are naturallyderived or synthetic and are active against a wide range ofmicroorganisms, such as bacteria, virus, yeast, parasite and fungi,and they may also exhibit an anti-tumor activity [46,47]. Severalfamilies of shrimp AMPs, such as penaeidins, lysozymes, crustins,ALFs and stylicins, have been identified and characterized [48,49].They are produced by and stored in the hemocytes, key cells in theshrimp immune system [50]. The recombinant proteins orsynthetic peptides of shrimp AMP family members have beentested in vitro for their antimicrobial activities against variousmicroorganisms (Table 2), where it is clear that different typesand/or isoforms exhibit a different but diverse spectrum of activ-ities and specificities.

Penaeidins, a unique family of AMPs specific to shrimps, havebeen isolated in some nine penaeid shrimp species, namely fromL. vannamei, Litopenaeus setiferus, M. japonicus, F. chinensis, Farfen-tepenaeus paulensis, Litopenaeus schmitti, Litopenaeus stylirostris,Fenneropeneaus indicus and P. monodon [49,51e53]. The signaturesof penaeidins are an unconstrained proline-rich domain (PRD) atthe N-terminal domain and six cysteine residues at the C-terminaldomain that form three disulfide linkages.

Penaeidins exhibit antifungal and anti-Gram-positive bacterialproperties. However, different classes display different target speci-ficities as a result of their sequence variability [49]. Apart from beingan AMP, penaeidin also displays an immunomodulation role asa cytokine that promotes the integrin-mediated adhesion of gran-ulocytes and semi-granulocytes in vitro [54]. Hemocytes have beenshown to producemore penaeidin at the site ofwound tissue but theaccumulation of hemocytes at the wound site is inhibited in penaei-din depleted (dsRNA knockdown) shrimps, but recovered afteradministration of recombinant penaeidin or its proline-rich domain


(PRD), suggesting that penaeidin acts as a pro-inflammatory cytokinein the wound-induced inflammation response of shrimp [55].

Crustin, a cationic peptide containing a cysteine-rich region anda whey acidic protein (WAP) domain, is another member of AMPsthat is unique to the Crustacea, being found in crabs, shrimps andseveral other crustaceans [49]. In shrimps, crustins have been iso-lated from L. vannamei and L. setiferus,M. japonicus, F. chinensis andP. monodon. Most shrimp crustins are Type II crustins that displaya strong antimicrobial activity against Gram-positive but not Gram-negative bacteria. However, an exception to this CrustinPm7, whichhas a strong antibacterial activity against both Gram-positive andGram-negative bacteria, including V. harveyi [49,56]. Other thanantibacterial activity, crustinPm1 and crustinPm7 are able toagglutinate bacterial cells and thus might be important for theirantibacterial action [57].

ALFs are antimicrobial peptides that have been identified andcharacterized in many crustaceans, including horseshoe crabs,shrimps and crabs [58e61]. ALFs are amphipathic peptides thatcontain two-highly conserved-cysteine residues that form a stabledisulfide loop habouring a highly conserved cluster of positivelycharged (Lys and Arg) residues. The 3D structure revealed that theactivity of ALF depends on the positively charged cluster within thedisulfide loop, which is in accord with explaining their highlyconserved nature, and that the amphipathic disulfide loop corre-sponds to the binding site for the lipid A component of bacterialcells [62]. The binding of ALFs to LPS results in the neutralization ofLPS [62e64]. The antimicrobial activity of the shrimp ALFs(ALFPm3, LvALF) has been reported to be a broad antimicrobialactivity against bacteria and fungi [50,59]. The synthetic disulfideloops from ALFPm3 and ALFSp have been shown unequivocally tohave antimicrobial activity on their own but at a lower level thanthat for the whole peptide [50]. The M. japonicus ALF-like peptide(MjALF) exhibits LPS neutralizing activity in the Limulus ameabo-cyte lysate and induces nitric oxide production in the murinemacrophage cell line RAW264.7 [58]. Gene knockdown experi-ments have revealed the likely immune-related function of ALFs inshrimps, where LvALF1 from L. vannamei can protect shrimps fromthe bacteria Vibrio penaeicida and the fungus Fusarium oxysporum[59]. The knockdown of ALFPm3 gene levels suggested that thefunction of ALFPm3 is vital to the shrimp, whereas ALFPm6depleted shrimps survived but showed an increase in the cumu-lative mortality and a faster mortality rate upon V. harveyi or WSSVinfections [65].

Lysozyme (E.C. 3.2.1.17) is an enzyme that cleaves the b-1,4-glycosidic linkages between N-acetylmuramic acid and N-acetyl-glucosamine in PG (meurin), a key component in the cell wall ofboth Gram-positive and Gram-negative bacteria and so causes celllysis. Lysozymes have traditionally been categorized into the threemajor types of (i) chicken-type lysozyme (c-type), (ii) goose-typelysozyme (g-type) and (iii) invertebrate-type lysozyme (i-type),based on their structural, catalytic and immunological character-istics. In shrimps, only the c-type and i-type lysozymes have beenreported so far and the c-type lysozymes account for the majority.The c-type lysozymes from M. japonicus [66], L. vannamei [67],P. monodon [68,69], Fenneropenaeus merguiensis [70], L. stylirostris[71] and F. chinensis [72] have all beenwell characterized. However,only two i-type lysozymes have been reported, one each inP. monodon and L. vannamei [73,74].

As stated above, lysozymes lyse both Gram-positive and Gram-negative bacteria and show a strong inhibition against the shrimppathogens Vibrio alginolyticus and Vibrio parahemolyticus. Pre-treating shrimp with the lysozyme from L. stylirostris protectedthem against subsequent WSSV infection, whilst the injection oflysozymemodulated the cellular and humoral defensemechanismsafter being suppressed by WSSV [71]. Gene silencing of c-type


Table 2The range of activity of shrimp AMPs.

Family Isoform Antimicrobial activity Other activity References

Crustins CruFc Gram-positive bacteria [76]Fc-crus 2 Gram-positive bacteria [56]Fc-crus 3 Gram-positive bacteria [56]crustinPm1 Gram-positive bacteria Agglutination [57,77]crustinPm5 Gram-positive bacteria [78]crustinPm7 Gram-positive bacteria Gram-negative bacteria Agglutination [57,79]SWDFc Gram-positive bacteria; Gram-negative bacteria; Fungi Protease inhibitory

activity against subtilisinA and protein K

[80]

SWDPm Gram-positive bacteria Protease inhibitoryactivity against subtilisin A

[81]

CruslikeFc1 Gram-positive bacteria [76]Penaeidin LitvanPen2 Gram-positive bacteria; Fungi [82]

LitvanPen3 Gram-positive bacteria; Fungi [82]LitvanPen4 Gram-positive bacteria; Fungi [83]FenchiPen5 Gram-negative bacteria; Gram-positive bacteria; Fungi [84]PenmonPen Gram-positive bacteria [85]PenmonPen3 Gram-positive bacteria; Fungi Cytokine [54,82]PenmonPen5 Gram-positive bacteria; Fungi; virus [51,86]

Lysozyme P. vannamei Gram-negative bacteria [73]M. japonicus Gram-negative bacteria [65]F. chinensis Gram-positive bacteria; Gram-negative bacteria [71]P. monodon Gram-negative bacteria [67,72]F. merguiensis Gram-positive bacteria; Gram-negative bacteria [69]L stylirostris Gram-positive bacteria; Gram-negative bacteria [70,87]

Antilipopolysaccharidefactors

ALFPm2 Gram-positive bacteria; Gram-negative bacteria Tharntada et al.unpublished data

ALFPm3 Gram-positive bacteria; Gram-negative bacteria;Fungi; virus

LPS and LTA binding activity [50,88,89]

LsALF1 Virus [59]MjALF1 LPS neutralizing activity [58]


lysozyme in M. japonicus revealed its role in suppressing thegrowth of Gram-negative bacteria in the hemolymph [75].

Stylicins are the most recent AMP family characterized in thepenaeid shrimps, but only the stylicin from L. stylirostris (LsStylicin1)has so far been characterized [48]. It is the first anionic AMP iden-tified in penaeid shrimps that contains a PRD at the N-terminus anda cysteine-rich domain at the C-terminus. Searching against thesequences deposited in the public databases revealed that severalputative stylicin homologs have been found; two from L. stylirostris,two partial sequences from L. vannamei and one partial sequencefrom P. monodon. LsStylicin1 has been shown to exhibit a poorlyspecific antibacterial activity against Vibrio sp. via a bacteriostaticeffect at a low peptide concentration and an agglutination activity athigher peptide concentrations. In addition, it exhibits a strongantifungal activity against the shrimp fungal pathogen, F. oxysporum,although the mechanism of this activity remains unknown.

3.2. Clotting

The chitin exoskeleton of crustaceans is an important barrieragainst pathogens as well as for maintaining the hemolymph andtissue integrity from diffusion. However, it does get damaged andthen breaches its protective barrier properties. To prevent the lossof hemolymph upon such injury and the invasion of infectedmicroorganisms, the rapid blood coagulation system at the site ofinjury is a prominent immune mechanism. The shrimp coagulationis believed to rely on the formation of a clottable protein (CP)polymer that is catalyzed by the Ca2þ dependent covalent linkage ofthe large dimeric CP by transglutaminase (TG) into long chains. Inshrimps, TG has been found in P. monodon [90,91], F. chinensis [92],M. japonicus [91] and L. vannamei [93]. Hemolymph CPs have beenidentified in M. japonicus, L. vannamei [94], F. paulensis [95] andP. monodon [84,96].


In shrimps, at least two types of shrimp TG (STG I and STG II)encoded by different chromosomal loci exist. However, only STG IIwas characterized as a hemocyte TG involved in the coagulationresponse [90]. In the horseshoe crab, clot formation is linked withthe release of antimicrobial substances [97]. In M. japonicus, it wasrecently shown that TG silencing caused the down-regulation ofAMP genes such as crustin and lysozyme, suggesting that therelease of these AMPs may depend on the activation of the coag-ulation system, or mainly on the activity of TG [98].

3.3. Pattern recognition receptors (PRRs), or pattern recognitionproteins (PRPs)

The innate immune system recognizes invadingmicroorganismsthrough a limited number of germline-encoded PRRs [99]. PRRsbind to pathogen associated molecular patterns (PAMPs), such asLPS, PG and BGs, which are present on the surface of microorgan-isms [100]. Different PRRs recognize specific PAMPs and triggersignaling pathways of the immune responses, including phagocy-tosis, nodule formation, encapsulation and synthesis of AMPs[101,102]. Several penaeid shrimp PRRs, including LGBP, BG-bindingprotein (GBP) and lectin. GBP has been isolated from the hemocytecDNA library of P. monodon and found to be constitutively expressedin the hemocyte with no significant change in the gene transcriptlevel after challenge with curdlan or heat-killed bacteria [103].

In F. chinensis, the LGBP transcript was mainly synthesized inhemocytes but was up-regulated upon bacterial challenge, whilstthe protein was localized on the membrane of most hemocytes,indicating that it might play a key role in the recognition ofbacterial pathogens [104]. Additionally, the L. stylirostris LGBPtranscript was induced after WSSV infection, suggesting that LGBPmight play a role in shrimpeWSSV interactions [105]. Recently, thenative LGBPs from the hemocytes of two Brazilian shrimps,F. paulensis and L. schmitti, were shown to induce the agglutination



of fungal cells [106] and, along with the recombinant LGBP fromP. monodon (rPmLGBP), to bind BG and strongly enhance the acti-vation of the proPO system [8,106].

Lectins contain a carbohydrate recognition domain (CRD) thathas the ability to bind to specific carbohydrate sequences expressedon different cell surfaces that then, through their crosslinking, leadsto bacterial agglutination and opsonization via enhancing the rateof phagocytosis of the microorganisms by hemocytes. Wang andWang [107] have reviewed the diversity and known functions oflectins in shrimps. Seven groups of lectins have been identified sofar, namely the C-type, L-type, P-type, M-type and fibrinogen-likedomain lectins, plus the galectins and calnexin/calreticulin. Inpenaeid shrimps, the C-type lectins are the most studied [108e115], and the hepatopancreas is the main tissue that synthesizesshrimp lectins, followed by the hemocytes. Lectins fromM. japonicus, F. chinensis and P. monodon have been shown todisplay a broad spectrum of bacteria-agglutination activitiesagainst both Gram-negative and Gram-positive bacteria[109,114,116].

Several shrimp lectins were found to be up-regulated afterWSSV or YHV infections [110,117e119]. Additionally, the PmAVlectin from P. monodon revealed a strong antiviral activity in termsof inhibiting the Singapore grouper iridovirus-induced cytopathiceffects in a fish cell line [108]. A mannose-binding C-type lectin (C-type lectin 1) from the shrimp L. vannamei (LvCTL1) binds toenvelope proteins of WSSV and protects the shrimps from viralinfection, prolonging their survival [119]. Recently, the L. vannameiC-type lectin-like domain, LvCTLD, was demonstrated to stimulatehemocyte encapsulation when LvCTLD was coated on agarosebeads and this was followed by melanization 24 h after encapsu-lation, which is somewhat similar to that observed for PmLT[111,118]. Interestingly, the in vivo injection of rLvCTLD alone orwith rLvCTLD plus YHV revealed a significant elevation of pheno-loxidase (PO) activity in the hemolymph of the injected-shrimps.This indicates that lectins may act as host recognition moleculesand play a key role in the shrimp defense mechanism againstmicroorganisms and viruses at the site of infection and throughactivation of the proPO system [118].

3.4. Proteinases and proteinase inhibitors

Proteinases and their inhibitors are ubiquitous in all livingorganisms and play crucial roles in various biological and physio-logical processes. Proteinases are part of several proteolyticcascades that are key components of the innate immune system ofshrimps and serve important roles in several related biochemicalpathways, such as those involved in apoptosis and melanization.Protease inhibitors, on the other hand, precisely regulate thepathway in order to prevent excessive activation of cascades andprevent consequent damage to host tissue. Moreover, hostproteinase inhibitors also function in the inhibition and clearancethe proteases of invading microorganisms. In shrimps, severalproteinases and proteinase inhibitors have been reported that arelinked to the immune system.

Clip domain serine proteinases (clip-SPs) and their homologs(clip-SPHs) are members of a proteinase family that are involved inthe shrimp innate immunity [120,121]. The structural features ofclip-SPs are that they contain the clip domain at the aminoterminus and a SP domain that has a catalytic triad (H, D and S) atthe active site. The clip-SPHs have a glycine substitute of the criticalserine in the SP-like domain resulting in the loss of SP activity.Several shrimp clip- (or pseudoclip-) SPHs have been identified[122e124]. As to their function, rclip-SPHs display a cell adhesionactivity [122], whilst SPH516 specifically interacts with the metalion-binding (MIB) domain of the YHV, suggesting that it might play


an important role in viral responses [124]. PmMasSPH has beenshown to be a multifunctional immune effector, where its N-terminal region containing the glycine-rich repeats and the clipdomain have in vitro antimicrobial activity against Gram-positivebacteria, and the C-terminal SP-like domain alone mediateshemocyte adhesion and displays binding activity to the shrimppathogenic bacterium, V. harveyi, as well as to LPS [125]. InF. chinensis, FcSP, a compliment C1r/C1s-Uegf-Bmp1 (CUB) domain-containing SP (FcCUBSP), and FcSPHs, including a masquerade SPH(FcMas), and single domain-containing SPHs (FcSPH and FcSPH2),are produced in the hemocytes that infiltrated the gills and theexpression of all FcSPs and FcSPHs are up-regulated in response topathogen challenge [126,127].

In penaeid shrimps, three families of serine protease inhibitors,the Kazal-type serine proteinase inhibitors (KPIs), serpins, andalpha-2-macroglobulins (A2Ms), have been identified and charac-terized. The KPIs contain one or more Kazal domains and theinhibitory activity of each KPIs depends on the action of each Kazaldomain that acts as a substrate analogue of, and competitivelybinds to, the cognate proteinase(s) forming a relatively stableproteinaseeproteinase inhibitor complex. The P1 amino acidresidue in the reactive site loop and the amino acid residues inother contact positions dictate the potency of the binding as well asthe specificity of the KPI to its cognate proteinase. Due to theexistence of more than one Kazal domain in each KPI it is predictedthat each KPIsmay inhibit more than one proteinase. The inhibitoryactivities of the KPIs of various invertebrates, including shrimps,have been summarized by Rimphanitchayakit and Tassanakajon[128]. In P. monodon, the major KPI is a five-domain KPI that isdesignated SPIPm2, which in addition to the proteinase inhibitoryactivities also possesses bacteriostatic [129] and antiviral [130]activities.

A2M is a nonspecific and broad-spectrum protease inhibitorthat belongs to the thioester containing protein superfamily andhas been highly conserved throughout evolution. A2Ms have beendiscovered in penaeid shrimps, such as, P. monodon [131],L. vannamei [132], F. chinensis [133] and F. paulensis [134]. Thedifferent A2Ms have different orders of multimerization but in caseof shrimp, they are homodimers. Immunogold labeling of A2M inthe hemocytes of F. paulensis indicated that it was stored in thevesicles of the shrimp granular hemocytes [134]. Interestingly, theshrimp A2M expression level is altered upon bacterial, viral andfungal infections at either the transcriptional or the translationallevels [39,131,133e135] revealing its likely diverse functions inshrimp immunity. Typically, A2M inhibits the active targetproteinase by a trapping mechanism (for a review see: [136]).Functional analysis of the P. monodon A2M revealed that it can bindto syntenin, a cytosolic protein with diverse biological functions[137]. Also, A2M may facilitate the entry of the phagocytosis acti-vating protein (PAP) into phagocytic cells and increase the survivalrate of the shrimp after being infected by WSSV [138].

Serpin is a family of high structural conserved inhibitors thatact as suicide-like substrates [139]. The reactive center loop(RCL) on the C-terminus side contains a scissile bond residingbetween the P1 and P10 amino acid residues. The P1 residuedetermines the target specificity. Serpin acts as suicide substrateinserting the RCL into the active site of proteinase and inducesa conformation change to form a covalent serpineproteinasecomplex. In shrimp, a few serpins from F. chinensis and P. mon-odon have been identified and characterized. They are localizedin hemocytes and expressed in response to pathogen challenges[140e142]. However, only PmSERPIN8 has been shown to beable to inhibit the growth of the Gram-positive bacteriumBacillus subtilis and to inhibit the activation of shrimp proPOsystem [142].



3.5. Melanization

Melanization is an important immune defense component ofinsects as well as in crustaceans. Melanin synthesis is achieved bythe proPO-activating system which is an enzymatic cascadeinvolving several enzymes including the key enzyme, PO [143]. TheproPO-activating system is initiated by the recognition and bindingof the PRPs to microbial cell wall components, such as PGs, LPS andBGs. The complex then triggers the activation of the SP cascade thatconverts proPO to active PO by a limited proteolysis. Active POoxidizes phenols into quinones that can nonspecifically crosslinkneighboring molecules to form melanin.

To date several genes associated with the shrimp proPO-activating system have been identified and characterized (see thereview by Amparyup et al. in this special issue). The existence oftwo proPOs has been reported in P. monodon [144] as well as in L.vannamei [145], while in most other crustaceans only a singleproPO gene has been found. PmproPO1 and PmproPO2 from thehemocytes of P. monodon, were both found to play an importantrole in the shrimp proPO system and immune defenses [144].Moreover, two proPO-activating enzymes (PmPPAE1 and PmPPAE2)that carry out the proteolysis of the proPO precursor have beenreported [121]. Additionally, several clip-SPs have been identifiedfrom a variety of shrimp species [126,146e148].

The importance of the proPO system in defense against bacterialinfection has been demonstrated by dsRNA mediated gene knock-down, which resulted in a significant reduction of the total POactivity and increased susceptibility of the shrimp to bacterialinfection [25,121,144]. Recently, PmLGBP was demonstrated to bindLPS and BG and to activate the proPO system in P. monodon [10].

3.6. Apoptosis pathway

Apoptosis is a genetically regulated cell death program thateliminates leftover, damaged, or harmful cells (such as virallyinfected). It is important for the normal development of theembryo, homeostasis and metamorphosis, and also for immunedefense [149,150]. Apoptosis plays a key role in the animal defensemechanism against viral infection. Many viruses have evolvedvarious strategies to inhibit host cell apoptosis during virus infec-tion, thereby prolonging the host cell viability until sufficientprogeny viruses have been produced [151]. However, some virusesinduce apoptosis at the late stage of viral infection in order tofacilitate the spread of progeny virus to other cells [152].

In penaeid shrimps, it has been reported that the shrimp viralpathogens, WSSV and YHV, can induce apoptosis upon infection[153,154]. Caspases are the important effector molecules thatmediate the apoptotic process [153]. Suppression of the PjCaspasegene of M. japonicus prior to WSSV infection led to an inhibition ofapoptosis following WSSV infection and an increase in the WSSVcopy number [153]. Overexpression of the recombinant P. monodoncaspase (PmCaspase) in SF-9 cells caused apoptotic-like morpho-logical features of the cells and characteristic DNA ladders [155]. Inaddition, two envelope proteins, VP38 and VP41B, of WSSV wereshown to bind to the promoter of PjCaspase and repress or activatethe PjCaspase promoter activity, respectively [156]. These resultsindicate that apoptosis plays a key role in the antiviral defensemechanisms in shrimp. However, in WSSV-infected M. japonicusshrimp, a high mortality and incidence of apoptosis are notsupportive of apoptosis being a protective immune response in thisshrimp [157]. Thus apoptosis might rather be involved in shrimpdeath as a result of viral infection.

This contradiction, although it remains unclear, could beexplained by the conflict between the virus and the host cells overthe fate of infected cells. The virus needs to prevent apoptosis during


the early to mid-infection stages in order to maintain thebiochemical and physiological competency of the host cellmachinery for viral replication and packaging to form multiplevirions, whereas the death of the infected host cell is favored by theshrimp to prevent new virion formation and release and so limit andend the infection. However, once new virions are produced, thevirus may require or benefit from death of that cell to release thenew virions into other cells of that host, or the surroundingmedia toinfect new hosts. The large-scale apoptosis from systemic infection,although lethal to the host shrimp, is not detrimental to a patho-genic virus [140,158]. Thus, the high density aquaculture ofcommercial shrimp farmingwould be expected to select for virulentpathogens where new hosts are easily found allowing high hori-zontal transmission rates, whereas in the lower host density naturalenvironment virulence is typically selected against for more benignpathogens and, perhaps if available, for vertical transmission.

Inhibitors of apoptotic proteins have also been identified inshrimps, including the defender against apoptotic death 1 (DAD1),translationally controlled tumor protein (TCTP/fortilin) and theinhibitor of apoptosis protein (IAP). The expression of DAD1 tran-scripts in P. monodon were significantly decreased after YHVchallenge [154] indicating that DAD1 likely plays a key role in theinfectivity of YHV and subsequent shrimp mortality. The transcriptlevel of TCTP/fortilin was highly up-regulated in P. monodonhemocytes following infection and then down-regulated in mori-bund shrimp [159]. Additionally, in vitro overexpression of shrimpfortilin in cultured mammalian cells protected the cells from celldeath under certain apoptosis-inducing conditions. Therefore, it ispossible that shrimp fortilin could protect shrimp cells undercertain conditions from death, like mammalian fortilin, and indi-cated that shrimp and human fortilin potentially use a commoncellular death pathway, including that involved in the response toWSSV infection [160].

IAP can block the apoptosis process by inhibiting the activities ofcaspases [161]. When the recombinant P. monodon IAP protein(PmIAP) was heterologously expressed in vitro in Drosophila celllines it could block the apoptosis induced by the Drosophila Reaperprotein [162]. Moreover, depletion of the LvIAP gene in L. vannameiby dsRNA mediated gene knockdown led to a decrease in thenumber of circulating hemocytes, caused by their extensiveapoptosis. Accordingly, LvIAP is central to the regulation of shrimphemocyte apoptosis and so control of hemocyte development and/or circulating numbers [163].

3.7. Signal transduction pathway

At least several conserved signaling pathways are involved inthe immune responses. The essential roles of Toll, Immunodefi-ciency (IMD) and the Janus kinase-signal transducer and activatorof transcription (JAK/STAT) pathways in shrimp immunity havebeen reported, and the shrimp IMD pathway component has beenreviewed by Li and Xiang [164].

The Toll-like receptor (TLR)-mediated NF-kB pathway is essen-tial for defending against viruses in insects and mammals. Severalmembers of the putative shrimp Toll signaling cascade wereidentified. TLR have been isolated from L. vannamei (LvToll, LvToll2and LvToll3) [165], P. monodon [166], F. chinensis (FcToll) [167] andM. japonicus [168]. In L. vannamei, LvToll, LvToll2 and LvToll3 wereup-regulated after WSSV challenge [165], with LvToll and LvToll3being localized to the membrane and cytoplasm, whilst LvToll2 wasconfined to the cytoplasm. However, only LvToll2 could signifi-cantly activate the promoters of the NF-kB pathway-controlledAMP genes.

The central potential positive regulator of the Toll pathway,Pelle, from L. vannamei (LvPelle) was recently characterized and



found to associate with LvTRAF6 during TLR signal transduction. Byanalogy to other known systems, it is speculated that a TLR/MyD88/Tube/Pelle/TRAF6/NF-kB cascade may exist in shrimps for immunegene regulation. Other components of TLR pathways have beenreported from L. vannamei and F. chinensis, such as Dorsal (LvDorsaland FcDorsal) and putative Spätzle-like Toll ligands (FcSpz andLvSpz1e3). Furthermore, a homolog of the interleukin-1 receptorassociated kinase-4 (IRAK-4), a central signal transduction medi-ator of the TLR and Toll/interleukin-1 receptor (TIR) pathways, hasbeen discovered in P. monodon (PmIRAK-4) [169].

In shrimps, the limited data available on the JAK/STAT pathwayhas revealed that it plays a significant role in the shrimp antiviralresponse. It is targeted byWSSV and serves as a transcription factorthat enhances transcription of theWSSV immediate early gene (ie1)and so controls ie1 transcription [170]. In accord, the in vitrotranslocation of activated STAT from the cytoplasm to the nucleushas been established in primary cultures of lymphoid organ cells ofWSSV-infected shrimp [171].

3.8. Antioxidant enzymes

Free radicals or reactive oxygen species (ROS) are normallyproduced in all living organisms during normal aerobic metabo-lism. Several stress conditions, such as environmental stresses(temperature, hypoxia and pH), sudden shortage of oxygen, dietarytoxicants and biological stresses (including pathogen infection), canlead to an increased level of ROS production and so result inoxidative stress inside the cell [172,173]. The increase in ROS specieslevel can cause oxidative damage to important cellular

Fig. 1. A schematic model of the shrimp immune syste


macromolecules (lipids, proteins, carbohydrates and nucleotides)that are components of the membranes, cellular enzymes and DNA[174].

Nevertheless, ROS species, including superoxide anion (O2�)

hydroxyl radical (OH�) and hydrogen peroxide (H2O2) are animportant part of the immune defense system that is produced tohelp eliminate the invading microbes [175,176]. In order to controlROSproduction, either to limit its spatial locationor to limit the levelsproduced, the antioxidant enzymes either convert O2

� to H2O2 bysuperoxide dismutase (SOD), convert H2O2 to water and oxygen bycatalase (CAT), or use H2O2 to oxidize substrates by various peroxi-dases, including glutathione peroxidase (GPx) [177]. This enzymaticantioxidant system is supplemented by the presence of with smallmolecule non-enzymatic antioxidants, including reduced gluta-thione (GSH), vitamin E, vitaminA, vitamin C and ceruloplasmin, andthe enzymes that produce some of them (e.g. glutathione-S-transferase (GST) and glutathione reductase (GR)) [172].

In penaeid shrimps, several oxidant enzymes as well as non-enzymatic oxidants have been isolated from P. monodon,F. chinensis and F. indicus through EST analysis. Their expressionlevels or enzyme activities have been shown to change afterpathogen infection, but in a differential manner according to thepathogen. Bacterial infections (V. alginolyticus, V. anguillarum, Vibrionigripulchritudo and Photobacterium damsel) induced a significantincrease in the activities of SOD, GPx, selenium-GPx, GST, CAT andGSH [173,178,179]. In contrast, their activities plus that of GR weresignificantly decreased in WSSV-infected P. monodon, F. indicus andF. chinensis [172,180e182]. In addition, the significant increase inlipid peroxidase activity in the muscle, hemolymph, gill andhepatopancreas tissues following WSSV infection [172,181]

m. For abbreviations and explanation see the text.



indicated that membrane dynamics may play an important role inWSSV infection in shrimps.

4. Conclusions and perspective

Despite the lack of whole genome information, several immune-related molecules of penaeid shrimp have been identified andcharacterized mainly by EST, SSH, microarrays and proteomicsanalyses. Many more genes and their participation in shrimpdefense will be unveiled when the whole genome data becomeavailable in the next few years. At present, functional analysis ofsome putative genes such as AMPs, proPOs, proteinases and theirinhibitors, anti-apototic proteins, PRPs, Toll receptors etc. hasrevealed the importance of these molecules in responses againstmajor shrimp pathogens. Although, the permanent cell lines havenot been successfully developed in crustaceans, the primary cellcultures, in particular the hematopoietic cell cultures of the crayfishPacifastacus leniusculus have been used to investigate the functionof several genes in crustaceans including shrimps. Alternatively, theRNAi technology has offered a simple and powerful method tostudy the gene function in shrimp. The proteineprotein interactionstudy between shrimp and their pathogen proteins also providesuseful information for understanding hostepathogen interactionand pathogenesis of shrimp pathogens. The current knowledge inshrimp immunity has been summarized and the outline picturewas shown here in Fig. 1. These studies provide insights to theinnate immune systems of shrimp that eventually lead to a bettercontrol of disease outbreaks and development of disease resistanceshrimp for the benefit of aquaculture industry.

Acknowledgements

Wewould like to thank Prof. Ikuo Hirono, the Editor, who invitesus to write this review. Our study on immune-related molecules inthe shrimp Penaeus monodon was supported by Thailand NationalCenter for Genetic Engineering and Biotechnology (BIOTEC) and theHigher Education Research promotion and National ResearchUniversity Project of Thailand, Office of the Higher EducationCommission (FW643A). We also thank the support from Chula-longkorn University to our Center of Excellence under the Ratch-adaphisek Somphot Endowment Fund. We would like to thankDr. Robert Douglas John Butcher at the Publication Counseling Unit(PCU), Faculty of Science, Chulalongkorn University, for Englishlanguage corrections of this manuscript.

References

[1] Lightner DV. Virus diseases of farmed shrimp in the Western Hemisphere(the Americas): a review. J Invertebr Pathol 2011;106:110e30.

[2] Flegel TW. Historic emergence, impact and current status of shrimp patho-gens in Asia. J Invertebr Pathol 2012;110:166e73.

[3] Bachère E, Gueguen Y, Gonzalez M, de Lorgeril J, Garnier J, Romestand B.Insights into the anti-microbial defense of marine invertebrates: the penaeidshrimps and the oyster Crassostrea gigas. Immunol Rev 2004;198:149e68.

[4] Martin GG, Graves BL. Fine structure and classification of shrimp hemocytes.J Morphol 1985;185:339e48.

[5] Sritunyalucksana K, Söderhäll K. The proPO and clotting system in crusta-ceans. Aquaculture 2000;191:53e69.

[6] Yu XQ, Zhu YF, Ma C, Fabrick JA, Kanost MR. Pattern recognition proteins inManduca sexta plasma. Insect Biochem Mol Biol 2002;32:1287e93.

[7] Janeway Jr CA, Medzhitov R. Innate immune recognition. Annu Rev Immunol2002;20:197e216.

[8] Amparyup P, Sutthangkul J, Charoensapsri W, Tassanakajon A. Patternrecognition protein binds to lipopolysaccharide and beta-1,3-glucan andactivates shrimp prophenoloxidase system. J Biol Chem 2012;287:10060e9.

[9] Chow S, Dougherty W, Sandifer P. Meiotic chromosome complements andnuclear DNA contents of four species of shrimps of the genus Penaeus. J CrustBiol 1990;10:29e36.

[10] Alcivar-Warren A, Meehan-Meola D, Wang YP, Guo XM, Zhou LH, Xiang LH,et al. Isolation and mapping of telomeric pentanucleotide (TAACC)(n) repeats


of the pacific white leg shrimp, Penaeus vannamei, using fluorescence in situhybridization. Mar Biotechnol 2006;8:467e80.

[11] Tassanakajon A, Klinbunga S, Paunglarp N, Rimphanitchayakit V, Udomkit A,Jitrapakdee S, et al. Penaeusmonodon gene discovery project: the generation ofan EST collection and establishment of a database. Gene 2006;384:104e12.

[12] O’Leary NA, Trent 3rd HF, Robalino J, Peck ME, McKillen DJ, Gross PS. Analysisof multiple tissue-specific cDNA libraries from the Pacific white leg shrimp,Litopenaeus vannamei. Integr Comp Biol 2006;46:931e9.

[13] Xiang J, Wang B, Li F, Liu B, Zhou Y, Tong W. Generation and analysis of10,443 ESTs from cephalothorax of Fenneropenaeus chinensis. In: The 2ndinternational conference on Bioinformatics and Biomedical Engineering.Shanghai, China, 2008; p. 74e80.

[14] Leu JH, Chen SH, Wang YB, Chen YC, Su SY, Lin CY, et al. A review of the majorpenaeid shrimp EST studies and the construction of a shrimp transcriptomedatabase based on the ESTs from four penaeid shrimp. Mar Biotechnol 2011;13:608e21.

[15] Robalino J, Almeida JS, McKillen D, Colglazier J, Trent 3rd HF, Chen YA, et al.Insights into the immune transcriptome of the shrimp Litopenaeus vannamei:tissue-specific expression profiles and transcriptomic responses to immunechallenge. Physiol Genomics 2007;29:44e56.

[16] Leu JH, Chang CC, Wu JL, Hsu CW, Hirono I, Aoki T, et al. Comparative analysisof differentially expressed genes in normal and white spot syndrome virusinfected Penaeus monodon. BMC Genomics 2007;8:120.

[17] Aoki T, Wang HC, Unajak S, Santos MD, Kondo H, Hirono I. Microarrayanalyses of shrimp immune responses. Mar Biotechnol 2011;13:629e38.

[18] Wang B, Li FH, Dong B, Zhang XJ, Zhang CS, Xiang JH. Discovery of the genes inresponse to white spot syndrome virus (WSSV) infection in Fenneropenaeuschinensis through cDNA microarray. Mar Biotechnol 2006;8:491e500.

[19] Wang B, Li F, Luan W, Xie Y, Zhang C, Luo Z, et al. Comparison of geneexpression profiles of Fenneropenaeus chinensis challenged with WSSV andVibrio. Mar Biotechnol 2008;10:664e75.

[20] Veloso A, Warr GW, Browdy CL, Chapman RW. The transcriptomic responseto viral infection of two strains of shrimp (Litopenaeus vannamei). Dev CompImmunol 2011;35:241e6.

[21] Wongpanya R, Aoki T, Hirono I, Yasuik M, Tassanakajon A. Analysis of geneexpression in haemocytes of shrimp Penaeus monodon challenged with whitespot syndrome virus by cDNA microarray. Science Asia 2007;33:165e74.

[22] Pongsomboon S, Tang S, Boonda S, Aoki T, Hirono I, Tassanakajon A. A cDNAmicroarray approach for analyzing transcriptional changes in Penaeus mon-odon after infection by pathogens. Fish Shellfish Immunol 2011;30:439e46.

[23] Dhar AK, Dettori A, Roux MM, Klimpel KR, Read B. Identification of differ-entially expressed genes in shrimp (Penaeus stylirostris) infected with Whitespot syndrome virus by cDNA microarrays. Arch Virol 2003;148:2381e96.

[24] Fagutao FF, YasuikeM, Caipang CM, Kondo H, Hirono I, Takahashi Y, et al. Geneexpression profile of hemocytes of Kuruma shrimp, Marsupenaeus japonicusfollowing peptidoglycan stimulation. Mar Biotechnol 2008;10:731e40.

[25] Fagutao FF, Yasuike M, Santos MD, Ruangpan L, Sangrunggruang K,Tassanakajon A, et al. Differential gene expression in black tiger shrimp,Penaeus monodon, following administration of oxytetracycline and oxolinicacid. Dev Comp Immunol 2009;33:1088e92.

[26] de la Vega E, Degnan BM, Hall MR, Wilson KJ. Differential expression ofimmune-related genes and transposable elements in black tiger shrimp(Penaeus monodon) exposed to a range of environmental stressors. FishShellfish Immunol 2007;23:1072e88.

[27] Uawisetwathana U, Leelatanawit R, Klanchui A, Prommoon J, Klinbunga S,Karoonuthaisiri N. Insights into eyestalk ablation mechanism to induceovarian maturation in the black tiger shrimp. PLOS One 2011;6.

[28] Somboonwiwat K, Supungul P, Rimphanitchayakit V, Aoki T, Hirono I,Tassanakajon A. Differentially expressed genes in hemocytes of Vibrio harveyi-challenged shrimp Penaeus monodon. J Biochem Mol Biol 2006;39:26e36.

[29] Wu J, Lin Q, Lim TK, Liu T, Hew CL. White spot syndrome virus proteins anddifferentiallyexpressedhostproteins identifiedinshrimpepitheliumbyshotgunproteomics and cleavable isotope-coded affinity tag. J Virol 2007;81:11681e9.

[30] Zhang J, Li F, Jiang H, Yu Y, Liu C, Li S, et al. Proteomic analysis of differentiallyexpressed proteins in lymphoid organ of Fenneropenaeus chinensis responseto Vibrio anguillarum stimulation. Fish Shellfish Immunol 2010;29:186e94.

[31] Jiang H, Li F, Xie Y, Huang B, Zhang J, Zhang C, et al. Comparative proteomicprofiles of the hepatopancreas in Fenneropenaeus chinensis response tohypoxic stress. Proteomics 2009;9:3353e67.

[32] Silvestre F, Huynh TT, Bernard A, Dorts J, Dieu M, Raes M, et al. A differentialproteomic approach to assess the effects of chemotherapeutics andproduction management strategy on giant tiger shrimp Penaeus monodon.Comp Biochem Physiol Part D: Genomics Proteomics 2010;5:227e33.

[33] Chai YM, Yu SS, Zhao XF, Zhu Q, Wang JX. Comparative proteomic profiles ofthe hepatopancreas in Fenneropenaeus chinensis response to white spotsyndrome virus. Fish Shellfish Immunol 2010;29:480e6.

[34] Wang HC, Wang HC, Leu JH, Kou GH, Wang AH, Lo CF. Protein expressionprofiling of the shrimp cellular response to white spot syndrome virusinfection. Dev Comp Immunol 2007;31:672e86.

[35] Wang HC, Ko TP, Lee YM, Leu JH, Ho CH, Huang WP, et al. White spotsyndrome virus protein ICP11: a histone-binding DNA mimic that disruptsnucleosome assembly. Proc Natl Acad Sci U S A 2008;105:20758e63.

[36] Chongsatja PO, Bourchookarn A, Lo CF, Thongboonkerd V, Krittanai C. Proteo-mic analysis of differentially expressed proteins in Penaeus vannamei hemo-cytes upon Taura syndrome virus infection. Proteomics 2007;7:3592e601.



[37] Rattanarojpong T, Wang HC, Lo CF, Flegel TW. Analysis of differentlyexpressed proteins and transcripts in gills of Penaeus vannamei after yellowhead virus infection. Proteomics 2007;7:3809e14.

[38] Bourchookarn A, Havanapan PO, Thongboonkerd V, Krittanai C. Proteomicanalysis of altered proteins in lymphoid organ of yellow head virus infectedPenaeus monodon. Biochim Biophys Acta 2008;1784:504e11.

[39] Somboonwiwat K, Chaikeeratisak V, Wang HC, Fang Lo C, Tassanakajon A.Proteomic analysis of differentially expressed proteins in Penaeus monodonhemocytes after Vibrio harveyi infection. Proteome Sci 2010;8:39.

[40] Chaikeeratisak V, Somboonwiwat K, Wang HC, Lo CF, Tassanakajon A. Pro-teomic analysis of differentially expressed proteins in the lymphoid organ ofVibrio harveyi-infected Penaeus monodon. Mol Biol Rep 2012;39:6367e77.

[41] Senapin S, Phongdara A. Binding of shrimp cellular proteins to Taurasyndrome viral capsid proteins VP1, VP2 and VP3. Virus Res 2006;122:69e77.

[42] Busayarat N, Senapin S, Tonganunt M, Phiwsaiya K, Meemetta W, Unajak S,et al. Shrimp laminin receptor binds with capsid proteins of two additionalshrimp RNA viruses YHV and IMNV. Fish Shellfish Immunol 2011;31:66e72.

[43] Tonganunt M, Nupan B, Saengsakda M, Suklour S, Wanna W, Senapin S, et al.The role of Pm-fortilin in protecting shrimp from white spot syndrome virus(WSSV) infection. Fish Shellfish Immunol 2008;25:633e7.

[44] Sangsuriya P, Rojtinnakorn J, Senapin S, Flegel TW. Identification and char-acterization of Alix/AIP1 interacting proteins from the black tiger shrimp,Penaeus monodon. J Fish Dis 2010;33:571e81.

[45] Sangsuriya P, Senapin S, Huang WP, Lo CF, Flegel TW. Co-interactive DNA-binding between a novel, immunophilin-like shrimp protein and VP15nucleocapsid protein of white spot syndrome virus. PLOS One 2011;6:e25420.

[46] Hancock RE, Diamond G. The role of cationic antimicrobial peptides in innatehost defences. Trends Microbiol 2000;8:402e10.

[47] Krepstakies M, Lucifora J, Nagel CH, Zeisel MB, Holstermann B, Hohenberg H,et al. A new class of synthetic peptide inhibitors blocks attachment and entryof human pathogenic viruses. J Infect Dis 2012;205:1654e64.

[48] Rolland JL, Abdelouahab M, Dupont J, Lefevre F, Bachère E, Romestand B.Stylicins, a new family of antimicrobial peptides from the Pacific blue shrimpLitopenaeus stylirostris. Mol Immunol 2010;47:1269e77.

[49] Tassanakajon A, Amparyup P, Somboonwiwat K, Supungul P. Cationic anti-microbial peptides in penaeid shrimp. Mar Biotechnol 2010;12:487e505.

[50] Somboonwiwat K, Marcos M, Tassanakajon A, Klinbunga S, Aumelas A,Romestand B, et al. Recombinant expression and anti-microbial activity ofanti-lipopolysaccharide factor (ALF) from the black tiger shrimp Penaeusmonodon. Dev Comp Immunol 2005;29:841e51.

[51] Woramongkolchai N, Supungul P, Tassanakajon A. The possible role ofpenaeidin5 from the black tiger shrimp, Penaeus monodon, in protectionagainst viral infection. Dev Comp Immunol 2011;35:530e6.

[52] Shanthi S, Vaseeharan B. cDNA cloning, characterization and expressionanalysis of a novel antimicrobial peptide gene penaeidin-3 (Fi-Pen3) fromthe haemocytes of Indian white shrimp Fenneropenaeus indicus. MicrobiolRes 2012;167:127e34.

[53] Gueguen Y, Garnier J, Robert L, Lefranc MP, Mougenot I, de Lorgeril J, et al. Theshrimp antimicrobial peptide penaeidin database: sequence-based classifica-tion and recommended nomenclature. Dev Comp Immunol 2006;30:283e8.

[54] Li CY, Yan HY, Song YL. Tiger shrimp (Penaeus monodon) penaeidin possessescytokine features to promote integrin-mediated granulocyte and semi-granulocyte adhesion. Fish Shellfish Immunol 2010;28:1e9.

[55] Li CY, Song YL. Proline-rich domain of penaeidin molecule exhibits autocrinefeature by attracting penaeidin-positive granulocytes toward the wound-induced inflammatory site. Fish Shellfish Immunol 2010;29:1044e52.

[56] Sun C, Du XJ, Xu WT, Zhang HW, Zhao XF, Wang JX. Molecular cloning andcharacterization of three crustins from the Chinese white shrimp, Fenner-openaeus chinensis. Fish Shellfish Immunol 2010;28:517e24.

[57] Krusong K, Poolpipat P, Supungul P, Tassanakajon A. A comparative study ofantimicrobial properties of crustinPm1 and crustinPm7 from the black tigershrimp Penaeus monodon. Dev Comp Immunol 2012;36:208e15.

[58] Nagoshi H, Inagawa H, Morii K, Harada H, Kohchi C, Nishizawa T, et al.Cloning and characterization of a LPS-regulatory gene having an LPS bindingdomain in Kuruma prawn Marsupenaeus japonicus. Mol Immunol 2006;43:2061e9.

[59] de la Vega E, O’Leary NA, Shockey JE, Robalino J, Payne C, Browdy CL, et al.Anti-lipopolysaccharide factor in Litopenaeus vannamei (LvALF): a broadspectrum antimicrobial peptide essential for shrimp immunity againstbacterial and fungal infection. Mol Immunol 2008;45:1916e25.

[60] Imjongjirak C, Amparyup P, Tassanakajon A, Sittipraneed S. Anti-lipopolysaccharide factor (ALF) of mud crab Scylla paramamosain: molecularcloning, genomic organization and the antimicrobial activity of its syntheticLPS binding domain. Mol Immunol 2007;44:3195e203.

[61] Imjongjirak C, Amparyup P, Tassanakajon A. Molecular cloning, genomicorganization and antibacterial activity of a second isoform of anti-lipopolysaccharide factor (ALF) from the mud crab, Scylla paramamosain. FishShellfish Immunol 2011;30:58e66.

[62] Yang Y, Boze H, Chemardin P, Padilla A, Moulin G, Tassanakajon A, et al. NMRstructure of rALF-Pm3, an anti-lipopolysaccharide factor from shrimp: modelof the possible lipid A-binding site. Biopolymers 2009;91:207e20.

[63] Hoess A, Watson S, Siber GR, Liddington R. Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at1.5 A resolution. EMBO J 1993;12:3351e6.


[64] Pristovsek P, Feher K, Szilagyi L, Kidric J. Structure of a synthetic fragment of theLALFproteinwhenbound to lipopolysaccharide. JMedChem2005;48:1666e70.

[65] Ponprateep S, Tharntada S, Somboonwiwat K, Tassanakajon A. Genesilencing reveals a crucial role for anti-lipopolysaccharide factors fromPenaeus monodon in the protection against microbial infections. Fish Shell-fish Immunol 2012;32:26e34.

[66] Hikima S, Hikima J, Rojtinnakorn J, Hirono I, Aoki T. Characterization andfunction of Kuruma shrimp lysozyme possessing lytic activity against Vibriospecies. Gene 2003;316:187e95.

[67] Sotelo-Mundo RR, Islas-Osuna MA, de-la-Re-Vega E, Hernandez-Lopez J,Vargas-Albores F, Yepiz-Plascencia G. cDNA cloning of the lysozyme of thewhite shrimp Penaeus vannamei. Fish Shellfish Immunol 2003;15:325e31.

[68] Tyagi A, Khushiramani R, Karunasagar I, Karunasagar I. Antivibrio activity ofrecombinant lysozyme expressed from black tiger shrimp, Penaeus monodon.Aquaculture 2007;272:246e53.

[69] Ye X, Gao FY, Zheng QM, Bai JJ, Wang H, Lao HH, et al. Cloning and charac-terization of the tiger shrimp lysozyme. Mol Biol Rep 2009;36:1239e46.

[70] Mai WJ, Hu CQ. Molecular cloning, characterization, expression and anti-bacterial analysis of a lysozyme homologue from Fenneropenaeus mer-guiensis. Mol Biol Rep 2009;36:1587e95.

[71] Mai WJ, Wang WN. Protection of blue shrimp (Litopenaeus stylirostris)against the White Spot Syndrome Virus (WSSV) when injected with shrimplysozyme. Fish Shellfish Immunol 2010;28:727e33.

[72] Bu X, Du X, Zhou W, Zhao X, Wang J. Molecular cloning, recombinantexpression and characterization of lysozyme from Chinese shrimp Fenner-openaeus chinensis. Sheng Wu Gong Cheng Xue Bao 2008;24:723e32.

[73] Supungul P, Rimphanitchayakit V, Aoki T, Hirono I, Tassanakajon A. Molec-ular characterization and expression analysis of a C-type and two novelmuramidase-deficient i-type lysozymes from Penaeus monodon. Fish Shell-fish Immunol 2010;28:490e8.

[74] Peregrino-Uriarte AB, Muhlia-Almazan AT, Arvizu-Flores AA, Gomez-Anduro G, Gollas-Galvan T, Yepiz-Plascencia G, et al. Shrimp invertebratelysozyme i-lyz: gene structure, molecular model and response of c and ilysozymes to lipopolysaccharide (LPS). Fish Shellfish Immunol 2012;32:230e6.

[75] Kaizu A, Fagutao FF, Kondo H, Aoki T, Hirono I. Functional analysis of C-typelysozyme in penaeid shrimp. J Biol Chem 2012;286:44344e9.

[76] Zhang J, Li F, Wang Z, Xiang J. Cloning and recombinant expression ofa crustin-like gene from Chinese shrimp, Fenneropenaeus chinensis. J Bio-technol 2007;127:605e14.

[77] Supungul P, Tang S, Maneeruttanarungroj C, Rimphanitchayakit V, Hirono I,Aoki T, et al. Cloning, expression and antimicrobial activity of crustinPm1,a major isoform of crustin, from the black tiger shrimp Penaeus monodon.Dev Comp Immunol 2008;32:61e70.

[78] Vatanavicharn T, Supungul P, Puanglarp N, Yingvilasprasert W,Tassanakajon A. Genomic structure, expression pattern and functionalcharacterization of crustinPm5, a unique isoform of crustin from Penaeusmonodon. Comp Biochem Physiol B, Biochem Mol Biol 2009;153:244e52.

[79] Amparyup P, Kondo H, Hirono I, Aoki T, Tassanakajon A. Molecular cloning,genomic organization and recombinant expression of a crustin-like antimi-crobial peptide from black tiger shrimp Penaeus monodon. Mol Immunol2008;45:1085e93.

[80] JiaYP, SunYD,WangZH,WangQ,WangXW,ZhaoXF, et al. A singlewhey acidicprotein domain (SWD)-containing peptide from fleshy prawn with antimi-crobial and proteinase inhibitory activities. Aquaculture 2008;284:246e59.

[81] Amparyup P, Donpudsa S, Tassanakajon A. Shrimp single WAP domain(SWD)-containing protein exhibits proteinase inhibitory and antimicrobialactivities. Dev Comp Immunol 2008;32:1497e509.

[82] Destoumieux D, Bulet P, Strub JM, Van Dorsselaer A, Bachère E. Recombinantexpression and range of activity of penaeidins, antimicrobial peptides frompenaeid shrimp. Eur J Biochem 1999;266:335e46.

[83] Cuthbertson BJ, Büllesbach EE, Fievet J, Bachère E, Gross PS. A new class(penaeidin class 4) of antimicrobial peptides from the Atlantic white shrimp(Litopenaeus setiferus) exhibits target specificity and an independent proline-rich-domain function. Biochem J 2004;381:79e86.

[84] Kang CJ, Xue JF, Liu N, Zhao XF, Wang JX. Characterization and expression ofa new subfamily member of penaeidin antimicrobial peptides (penaeidin 5)from Fenneropenaeus chinensis. Mol Immunol 2007;44:1535e43.

[85] Ho SH, Chao YC, Tsao HW, Sakai M, Chou HN, Song YL. Molecular cloning andrecombinant expression of tiger shrimp Penaeus monodon penaeidin. FishPathol 2004;39:15e23.

[86] Hu SY, Huang JH, Huang WT, Yeh YH, Chen MHC, Gong HY, et al. Structureand function of antimicrobial peptide penaeidin-5 from the black tigershrimp Penaeus monodon. Aquaculture 2006;260:61e8.

[87] de Lorgeril J, Gueguen Y, Goarant C, Goyard E, Mugnier C, Fievet J, et al.A relationship between antimicrobial peptide gene expression and capacityof a selected shrimp line to survive a Vibrio infection. Mol Immunol 2008;45:3438e45.

[88] Somboonwiwat K, Bachère E, Rimphanitchayakit V, Tassanakajon A. Locali-zation of anti-lipopolysaccharide factor (ALFPm3) in tissues of the black tigershrimp, Penaeus monodon, and characterization of its binding properties. DevComp Immunol 2008;32:1170e6.

[89] Tharntada S, Ponprateep S, Somboonwiwat K, Liu H, Söderhäll I, Söderhäll K,et al. Role of anti-lipopolysaccharide factor from the black tiger shrimp,Penaeus monodon, in protection from white spot syndrome virus infection.J Gen Virol 2009;90:1491e8.



[90] Chen MY, Hu KY, Huang CC, Song YL. More than one type of trans-glutaminase in invertebrates? A second type of transglutaminase is involvedin shrimp coagulation. Dev Comp Immunol 2005;29:1003e16.

[91] Yeh MS, Kao LR, Huang CJ, Tsai IH. Biochemical characterization and cloningof transglutaminases responsible for hemolymph clotting in Penaeus mono-don andMarsupenaeus japonicus. Biochim Biophys Acta 2006;1764:1167e78.

[92] Liu YC, Li FH, Wang B, Dong B, Zhang QL, Luan W, et al. A transglutaminasefrom Chinese shrimp (Fenneropenaeus chinensis), full-length cDNA cloning,tissue localization and expression profile after challenge. Fish ShellfishImmunol 2007;22:576e88.

[93] Yeh MS, Liu CH, Hung CW, Cheng W. cDNA cloning, identification, tissuelocalisation, and transcription profile of a transglutaminase from whiteshrimp, Litopenaeus vannamei, after infection by Vibrio alginolyticus. FishShellfish Immunol 2009;27:748e56.

[94] Cheng W, Tsai IH, Huang CJ, Chiang PC, Cheng CH, Yeh MS. Cloning andcharacterization of hemolymph clottable proteins of Kuruma prawn (Mar-supenaeus japonicus) and white shrimp (Litopenaeus vannamei). Dev CompImmunol 2008;32:265e74.

[95] Perazzolo LM, Lorenzini DM, Daffre S, Barracco MA. Purification and partialcharacterization of the plasma clotting protein from the pink shrimp Far-fantepenaeus paulensis. Comp Biochem Physiol B, Biochem Mol Biol 2005;142:302e7.

[96] Yeh MS, Huang CJ, Leu JH, Lee YC, Tsai IH. Molecular cloning and charac-terization of a hemolymph clottable protein from tiger shrimp (Penaeusmonodon). Eur J Biochem 1999;266:624e33.

[97] Iwanaga S, Lee BL. Recent advances in the innate immunity of invertebrateanimals. J Biochem Mol Biol 2005;38:128e50.

[98] Fagutao FE, Maningas MBB, Kondo H, Aoki T, Hirono I. Transglutaminase regu-lates immune-related genes in shrimp. Fish Shellfish Immunol 2012;32:711e5.

[99] Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity.Cell 2006;124:783e801.

[100] Medzhitov R, Janeway C. Innate immune recognition: mechanisms andpathways. Immunol Rev 2000;173:89e97.

[101] Gillespie JP, Kanost MR, Trenczek T. Biological mediators of insect immunity.Annu Rev Entomol 1997;42:611e43.

[102] Yu XQ, Kanost MR. Binding of hemolin to bacterial lipopolysaccharide andlipoteichoic acid. An immunoglobulin superfamily member from insects asa pattern-recognition receptor. Eur J Biochem 2002;269:1827e34.

[103] Sritunyalucksana K, Lee SY, Söderhäll K. A beta-1,3-glucan binding proteinfrom the black tiger shrimp, Penaeus monodon. Dev Comp Immunol 2002;26:237e45.

[104] Du XJ, Zhao XF, Wang JX. Molecular cloning and characterization of a lipo-polysaccharide and b-1,3-glucan binding protein from fleshy prawn(Fenenropenaeus chinensis). Mol Immunol 2007;44:1085e94.

[105] Roux MM, Pain A, Klimpel KR, Dhar AK. The lipopolysaccharide and beta-1,3-glucan binding protein gene is upregulated in white spot virus-infectedshrimp (Penaeus stylirostris). J Virol 2002;76:7140e9.

[106] Goncalves P, Vernal J, Rosa RD, Yepiz-Plascencia G, de Souza CR, Barracco MA,et al. Evidence for a novel biological role for the multifunctional beta-1,3-glucan binding protein in shrimp. Mol Immunol 2012;51:363e7.

[107] Wang XW, Wang JX. Diversity and multiple functions of lectins in shrimpimmunity. Dev Comp Immunol http://dx.doi.org/10.1016/j.dci.2012.04.009.

[108] Luo T, Zhang X, Shao Z, Xu X. PmAV, a novel gene involved in virus resistanceof shrimp Penaeus monodon. FEBS Lett 2003;551:53e7.

[109] Luo T, Yang H, Li F, Zhang X, Xu X. Purification, characterization and cDNAcloning of a novel lipopolysaccharide-binding lectin from the shrimpPenaeus monodon. Dev Comp Immunol 2006;30:607e17.

[110] Ma TH, Tiu SH, He JG, Chan SM. Molecular cloning of a C-type lectin (LvLT)from the shrimp Litopenaeus vannamei: early gene down-regulation afterWSSV infection. Fish Shellfish Immunol 2007;23:430e7.

[111] Ma THT, Benzie JAH, He JG, Chan SM. PmLT, a C-type lectin specific tohepatopancreas is involved in the innate defense of the shrimp Penaeusmonodon. J Invertebr Pathol 2008;99:332e41.

[112] Liu YC, Li FH, Dong B, Wang B, LuanW, Zhang XJ, et al. Molecular cloning, char-acterization and expression analysis of a putative C-type lectin (Fclectin) gene inChinese shrimp Fenneropenaeus chinensis. Mol Immunol 2007;44:598e607.

[113] Song KK, Li DF, Zhang MC, Yang HJ, Ruan LW, Xu X. Cloning and character-ization of three novel WSSV recognizing lectins from shrimp Marsupenaeusjaponicus. Fish Shellfish Immunol 2010;28:596e603.

[114] Yang H, Luo T, Li F, Li S, Xu X. Purification and characterisation of a calcium-independent lectin (PjLec) from the haemolymph of the shrimp Penaeusjaponicus. Fish Shellfish Immunol 2007;22:88e97.

[115] Wei X, Liu X, Yang J, Fang J, Qiao H, Zhang Y, et al. Two C-type lectins fromshrimp Litopenaeus vannamei that might be involved in immune responseagainst bacteria and virus. Fish Shellfish Immunol 2012;32:132e40.

[116] Wang XW, Zhang XW, Xu WT, Zhao XF, Wang JX. A novel C-type lectin(FcLec4) facilitates the clearance of Vibrio anguillarum in vivo in Chinesewhite shrimp. Dev Comp Immunol 2009;33:1039e47.

[117] Wang XW, Xu WT, Zhang XW, Zhao XF, Yu XQ, Wang JX. A C-type lectin isinvolved in the innate immune response of Chinese white shrimp. FishShellfish Immunol 2009;27:556e62.

[118] Junkunlo K, Prachumwat A, Tangprasittipap A, Senapin S, Borwornpinyo S,Flegel TW, et al. A novel lectin domain-containing protein (LvCTLD) associ-ated with response of the white leg shrimp Penaeus (Litopenaeus) vannameito yellow head virus (YHV). Dev Comp Immunol 2012;37:334e41.


[119] Zhao ZY, Yin ZX, Xu XP, Weng SP, Rao XY, Dai ZX, et al. A novel C-type lectinfrom the shrimp Litopenaeus vannamei possesses anti-white spot syndromevirus activity. J Virol 2009;83:347e56.

[120] Jiang H, Kanost MR. The clip-domain family of serine proteinases inarthropods. Insect Biochem Mol Biol 2000;30:95e105.

[121] Charoensapsri W, Amparyup P, Hirono I, Aoki T, Tassanakajon A. Genesilencing of a prophenoloxidase activating enzyme in the shrimp, Penaeusmonodon, increases susceptibility to Vibrio harveyi infection. Dev CompImmunol 2009;33:811e20.

[122] Lin CY, Hu KY, Ho SH, Song YL. Cloning and characterization of a shrimp clipdomain serine protease homolog (c-SPH) as a cell adhesion molecule. DevComp Immunol 2006;30:1132e44.

[123] Amparyup P, Jitvaropas R, Pulsook N, Tassanakajon A. Molecular cloning,characterization and expression of a masquerade-like serine proteinasehomologue from black tiger shrimp Penaeus monodon. Fish ShellfishImmunol 2007;22:535e46.

[124] Sriphaijit T, Flegel TW, Senapin S. Characterization of a shrimp serineprotease homolog, a binding protein of yellow head virus. Dev CompImmunol 2007;31:1145e58.

[125] Jitvaropas R, Amparyup P, Gross PS, Tassanakajon A. Functional character-ization of a masquerade-like serine proteinase homologue from the blacktiger shrimp Penaeus monodon. Comp Biochem Physiol B, Biochem Mol Biol2009;153:236e43.

[126] Ren Q, Xu ZL, Wang XW, Zhao XF, Wang JX. Clip domain serine protease andits homolog respond to Vibrio challenge in Chinese white shrimp, Fenner-openaeus chinensis. Fish Shellfish Immunol 2009;26:787e98.

[127] Ren Q, Zhao XF, Wang JX. Identification of three different types of serineproteases (one SP and two SPHs) in Chinese white shrimp. Fish ShellfishImmunol 2011;30:456e66.

[128] Rimphanitchayakit V, Tassanakajon A. Structure and function of invertebrateKazal-type serine proteinase inhibitors. Dev Comp Immunol 2010;34:377e86.

[129] Donpudsa S, Tassanakajon A, Rimphanitchayakit V. Domain inhibitory andbacteriostatic activities of the five-domain Kazal-type serine proteinaseinhibitor from black tiger shrimp Penaeus monodon. Dev Comp Immunol2009;33:481e8.

[130] Ponprateep S, Tassanakajon A, Rimphanitchayakit V. A Kazal type serineproteinase SPIPm2 from the black tiger shrimp Penaeus monodon is capableof neutralization and protection of hemocytes from the white spot syndromevirus. Fish Shellfish Immunol 2011;31:1179e85.

[131] Lin YC, Vaseeharan B, Ko CF, Chiou TT, Chen JC. Molecular cloning and charac-terisation of a proteinase inhibitor, alpha 2-macroglobulin (alpha2-M) from thehaemocytes of tiger shrimpPenaeusmonodon.Mol Immunol 2007;44:1065e74.

[132] Lin YC, Vaseeharan B, Chen JC. Molecular cloning and phylogenetic analysison alpha2-macroglobulin (alpha2-M) of white shrimp Litopenaeus vannamei.Dev Comp Immunol 2008;32:317e29.

[133] Ma H, Wang B, Zhang J, Li F, Xiang J. Multiple forms of alpha-2 macroglobulinin shrimp Fenneropenaeus chinesis and their transcriptional response toWSSV or Vibrio pathogen infection. Dev Comp Immunol 2010;34:677e84.

[134] Perazzolo LM, Bachère E, Rosa RD, Goncalves P, Andreatta ER, Daffre S, et al.Alpha2-macroglobulin from an Atlantic shrimp: biochemical characteriza-tion, sub-cellular localization and gene expression upon fungal challenge.Fish Shellfish Immunol 2011;31:938e43.

[135] Ho PY, Cheng CH, Cheng W. Identification and cloning of the alpha2-macroglobulin of giant freshwater prawn Macrobrachium rosenbergii andits expression in relation with the molt stage and bacteria injection. FishShellfish Immunol 2009;26:459e66.

[136] Armstrong PB. Role of a2-macroglobulin in the immune responses ofinvertebrates. Invert Surviv J 2010;7:165e80.

[137] Tonganunt M, Phongdara A, Chotigeat W, Fujise K. Identification and char-acterization of syntenin binding protein in the black tiger shrimp Penaeusmonodon. J Biotechnol 2005;120:135e45.

[138] Chotigeat W, Deachamag P, Phongdara A. Identification of a protein bindingto the phagocytosis activating protein (PAP) in immunized black tigershrimp. Aquaculture 2007;271:112e20.

[139] Huntington JA. Serpin structure, function and dysfunction. J Thromb Hae-most 2011;9(Suppl. 1):26e34.

[140] Liu HP, Söderhäll K, Jiravanichpaisal P. Antiviral immunity in crustaceans.Fish Shellfish Immunol 2009;27:79e88.

[141] Homvises T, Tassanakajon A, Somboonwiwat K. Penaeus monodon SERPIN,PmSERPIN6, is implicated in the shrimp innate immunity. Fish ShellfishImmunol 2010;29:890e8.

[142] Somnuk S, Tassanakajon A, Rimphanitchayakit V. Gene expression andcharacterization of a serine proteinase inhibitor PmSERPIN8 from the blacktiger shrimp Penaeus monodon. Fish Shellfish Immunol 2012;33:332e41.

[143] Gorman MJ, Dittmer NT, Marshall JL, Kanost MR. Characterization of themulticopper oxidase gene family in Anopheles gambiae. Insect Biochem MolBiol 2008;38:817e24.

[144] Amparyup P, Charoensapsri W, Tassanakajon A. Two prophenoloxidases areimportant for the survival of Vibrio harveyi challenged shrimp Penaeusmonodon. Dev Comp Immunol 2009;33:247e56.

[145] Ai HS, Liao JX, Huang XD, Yin ZX, Weng SP, Zhao ZY, et al. A novelprophenoloxidase 2 exists in shrimp hemocytes. Dev Comp Immunol 2009;33:59e68.

[146] Amparyup P, Wiriyaukaradecha K, Charoensapsri W, Tassanakajon A. A clipdomain serine proteinase plays a role in antibacterial defense but is not



required for prophenoloxidase activation in shrimp. Dev Comp Immunol2010;34:168e76.

[147] Jang IK, Pang Z, Yu J, Kim SK, Seo HC, Cho YR. Selectively enhancedexpression of prophenoloxidase activating enzyme 1 (PPAE1) at a bacteriaclearance site in the white shrimp, Litopenaeus vannamei. BMC Immunol2011;12:70.

[148] Vaseeharan B, Shanthi S, Prabhu NM. A novel clip domain serine proteinase(SPs) gene from the haemocytes of Indian white shrimp Fenneropenaeusindicus: molecular cloning, characterization and expression analysis. FishShellfish Immunol 2011;30:980e5.

[149] Wen R, Li F, Xie Y, Li S, Xiang J. A homolog of the cell apoptosis susceptibilitygene involved in ovary development of Chinese shrimp Fenneropenaeuschinensis. Biol Reprod 2012;86:1e7.

[150] Portt L, Norman G, Clapp C, Greenwood M, Greenwood MT. Anti-apoptosisand cell survival: a review. Biochim Biophys Acta 2011;1813:238e59.

[151] Koyama AH, Fukumori T, Fujita M, Irie H, Adachi A. Physiological significanceof apoptosis in animal virus infection. Microbes Infect 2000;2:1111e7.

[152] Best SM. Viral subversion of apoptotic enzymes: escape from death row.Annu Rev Microbiol 2008;62:171e92.

[153] Wang L, Zhi B, Wu W, Zhang X. Requirement for shrimp caspase in apoptosisagainst virus infection. Dev Comp Immunol 2008;32:706e15.

[154] Molthathong S, Senapin S, Klinbunga S, Puanglarp N, Rojtinnakorn J,Flegel TW. Down-regulation of defender against apoptotic death (DAD1)after yellow head virus (YHV) challenge in black tiger shrimp Penaeusmonodon. Fish Shellfish Immunol 2008;24:173e9.

[155] Leu JH, Wang HC, Kou GH, Lo CF. Penaeus monodon caspase is targeted bya white spot syndrome virus anti-apoptosis protein. Dev Comp Immunol2008;32:476e86.

[156] Zuo H, Chen C, Gao Y, Lin J, Jin C, Wang W. Regulation of shrimp PjCaspasepromoter activity by WSSV VP38 and VP41B. Fish Shellfish Immunol 2011;30:1188e91.

[157] Wu JL, Muroga K. Apoptosis does not play an important role in the resistanceof ‘immune’ Penaeus japonicas against white spot syndrome virus. J Fish Dis2004;27:15e21.

[158] Flegel TW, Sritunyalucksana K. Shrimp molecular responses to viral patho-gens. Mar Biotechnol 2011;13:587e607.

[159] Bangrak P, Graidist P, Chotigeat W, Phongdara A. Molecular cloning andexpression of a mammalian homologue of a translationally controlled tumorprotein (TCTP) gene from Penaeus monodon shrimp. J Biotechnol 2004;108:219e26.

[160] Graidist P, Fujise K, Wanna W, Sritunyalucksana K, Phongdara A. Establishinga role for shrimp fortilin in preventing cell death. Aquaculture 2006;255:157e64.

[161] Deveraux QL, Reed JC. IAP family proteins-suppressors of apoptosis. GenesDev 1999;13:239e52.

[162] Leu JH, Kuo YC, Kou GH, Lo CF. Molecular cloning and characterization of aninhibitor of apoptosis protein (IAP) from the tiger shrimp, Penaeus monodon.Dev Comp Immunol 2008;32:121e33.

[163] Leu JH, Chen YC, Chen LL, Chen KY, Huang HT, Ho JM, et al. Litopenaeusvannamei inhibitor of apoptosis protein 1 (LvIAP1) is essential for shrimpsurvival. Dev Comp Immunol 2012;38:78e87.

[164] Li F, Xiang J. Recent advances in researches on the innate immunity of shrimpin China. Dev Comp Immunol http://dx.doi.org/10.1016/j.bbr.2011.03.031.

[165] Wang PH, Liang JP, Gu ZH, Wan DH, Weng SP, Yu XQ, et al. Molecular cloning,characterization and expression analysis of two novel Tolls (LvToll2 andLvToll3) and three putative Spatzle-like Toll ligands (LvSpz1-3) from Lito-penaeus vannamei. Dev Comp Immunol 2012;36:359e71.


[166] Assavalapsakul W, Panyim S. Molecular cloning and tissue distribution of theToll receptor in the black tiger shrimp, Penaeus monodon. Genet Mol Res2012;11:484e93.

[167] Yang C, Zhang J, Li F, Ma H, Zhang Q, Jose Priya TA, et al. A Toll receptor fromChinese shrimp Fenneropenaeus chinensis is responsive to Vibrio anguillaruminfection. Fish Shellfish Immunol 2008;24:564e74.

[168] Mekata T, Kono T, Yoshida T, Sakai M, Itami T. Identification of cDNAencoding Toll receptor, MjToll gene from Kuruma shrimp, Marsupenaeusjaponicus. Fish Shellfish Immunol 2008;24:122e33.

[169] Watthanasurorot A, Söderhäll K, Jiravanichpaisal PA. Mammalian likeinterleukin-1 receptor-associated kinase 4 (IRAK-4), a TIR signaling mediatorin intestinal innate immunity of black tiger shrimp (Penaeus monodon).Biochem Biophys Res Commun 2012;417:623e9.

[170] Liu WJ, Chang YS, Wang AHJ, Kou GH, Lo CF. White spot syndrome virusannexes a shrimp STAT to enhance expression of the immediate-early geneie1. J Virol 2007;81:1461e71.

[171] Chen WY, Ho KC, Leu JH, Liu KF, Wang HC, Kou GH, et al. WSSV infectionactivates STAT in shrimp. Dev Comp Immunol 2008;32:1142e50.

[172] Mohankumar K, Ramasamy P. White spot syndrome virus infectiondecreases the activity of antioxidant enzymes in Fenneropenaeus indicus.Virus Res 2006;115:69e75.

[173] Castex M, Lemaire P, Wabete N, Chim L. Effect of probiotic Pediococcusacidilactici on antioxidant defences and oxidative stress of Litopenaeus styl-irostris under Vibrio nigripulchritudo challenge. Fish Shellfish Immunol 2010;28:622e31.

[174] Chang CC, Slesak I, Jorda L, Sotnikov A, Melzer M, Miszalski Z, et al. Arabi-dopsis chloroplastic glutathione peroxidases play a role in cross talkbetween photooxidative stress and immune responses. Plant Physiol 2009;150:670e83.

[175] Bachère E, Mialhe E, Noel D, Boulo V, Morvan A, Rodriguez J. Knowledge andresearch prospects in marine mollusk and crustacean immunology. Aqua-culture 1995;132:17e32.

[176] Munoz M, Cedeno R, Rodriguez J, van der Knaap WPW, Mialhe E,Bachère E. Measurement of reactive oxygen intermediate production inhaemocytes of the penaeid shrimp, Penaeus vannamei. Aquaculture 2000;191:89e107.

[177] Abele D, Puntarulo S. Formation of reactive species and induction of anti-oxidant defence systems in polar and temperate marine invertebrates andfish. Comp Biochem Physiol A 2004;138:405e15.

[178] Liu KF, Yeh MS, Kou GH, Cheng W, Lo CF. Identification and cloning ofa selenium-dependent glutathione peroxidase from tiger shrimp, Penaeusmonodon, and its transcription following pathogen infection and related tothe molt stages. Dev Comp Immunol 2010;34:935e44.

[179] Ren Q, Sun RR, Zhao XF, Wang JX. A selenium-dependent glutathioneperoxidase (Se-GPx) and two glutathione S-transferases (GSTs) from Chineseshrimp (Fenneropenaeus chinensis). Comp Biochem Physiol C Toxicol Phar-macol 2009;149:613e23.

[180] Mathew S, Kumar KA, Anandan R, ViswanathanNair PG, Devadasan K. Changesin tissuedefence system inwhite spot syndromevirus (WSSV) infected Penaeusmonodon. Comp Biochem Physiol C Toxicol Pharmacol 2007;145:315e20.

[181] Rameshthangam P, Ramasamy P. Antioxidant and membrane boundenzymes activity in WSSV-infected Penaeus monodon fabricius. Aquaculture2006;254:32e9.

[182] Zhang Q, Li F, Wang B, Zhang J, Liu Y, Zhou Q, et al. The mitochondrialmanganese superoxide dismutase gene in Chinese shrimp Fenneropenaeuschinensis: cloning, distribution and expression. Dev Comp Immunol 2007;31:429e40.


Fish & Shellﬁsh Immunology - GBC - Global Biotech ...€¦ · aCenter of Excellence for Molecular...

Documents

Transcript of Fish & Shellﬁsh Immunology - GBC - Global Biotech ...€¦ · aCenter of Excellence for Molecular...