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Fish & Shellfish Immunology 34 (2013) 990e1001

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Fish & Shellfish Immunology

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Prophenoloxidase system and its role in shrimp immune responses against majorpathogens

Piti Amparyup a,b,*, Walaiporn Charoensapsri a, Anchalee Tassanakajon a

aCenter of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, ThailandbNational Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Paholyothin Road, Klong1, Klong Luang,Pathumthani 12120, Thailand

a r t i c l e i n f o

Article history:Received 28 May 2012Received in revised form8 August 2012Accepted 17 August 2012Available online 28 August 2012

Keywords:ShrimpPenaeusPhenoloxidaseMelanizationproPO system

* Corresponding author. National Center for Genenology (BIOTEC), National Science and Technology D113 Paholyothin Road, Klong1, Klong Luang, Pathumfax: þ66 2 218 5414.

E-mail address: piti.amp@biotec.or.th (P. Amparyu

1050-4648/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.fsi.2012.08.019

a b s t r a c t

The global shrimp industry still faces various serious disease-related problems that are mainly caused bypathogenic bacteria and viruses. Understanding the host defense mechanisms is likely to be beneficial indesigning and implementing effective strategies to solve the current and future pathogen-relatedproblems. Melanization, which is performed by phenoloxidase (PO) and controlled by the proph-enoloxidase (proPO) activation cascade, plays an important role in the invertebrate immune system inallowing a rapid response to pathogen infection. The activation of the proPO system, by the specificrecognition of microorganisms by pattern-recognition proteins (PRPs), triggers a serine proteinasecascade, eventually leading to the cleavage of the inactive proPO to the active PO that functions toproduce the melanin and toxic reactive intermediates against invading pathogens. This review highlightsthe recent discoveries of the critical roles of the proPO system in the shrimp immune responses againstmajor pathogens, and emphasizes the functional characterizations of four major groups of genes andproteins in the proPO cascade in penaeid shrimp, that is the PRPs, serine proteinases, proPO andinhibitors.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Successful mass monoculture shrimp production, with itsinherent high shrimp density rearing, requires the use of effectivedisease prevention strategies, for which a good understanding ofthe basic immune functions is invaluable [1].

Invertebrates survive microbial infections in the absence of anypre-existing highly evolved adaptive immunity. Instead theyinitially rely on a fixed non-specific immunemechanism to preventpathogen entry and spread that involves structural barriers toprevent pathogen entry, such as a hard cuticle, tegumental glands,branchial podocytes, epithelial immunity, autonomy and regener-ation of appendages and rapid would healing to prevent loss ofhemolymph and seal the would. However, should their mecha-nisms fail they mount an immediate multiple innate immuneresponse to efficiently defend themselves against the onset ofpathogens [2]. The mechanism of the innate immune system in

tic Engineering and Biotech-evelopment Agency (NSTDA),thani 12120, Thailand. Tel./

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response to the inevitably encounter pathogens requires therecognition of the evolutionarily conserved microbe-specificmolecules, known as pathogen-associated molecular patterns(PAMPs) by a set of well defined receptors referred to as pattern- orpathogen-recognition receptors (PRPs) [2e5]. Recognition of theunique patterns of PAMPs eventually results in the widespreadinnate immune activation, including via the release of solublemolecules and the circulating hemocyte mediated humoral andcellular components [3,6].

The mobile non-specific immune system has two principalcomponents, the humoral and cellular systems, which are bothactivated upon immune challenge and work together. The cellularcomponent involves those mediated by hemocytes and thehumoral components involve the cell free components of thehemolymph. However, note that as stated already they are notindependent of each other but rather are interactive and interde-pendent and so function synergistically to protect the shrimp andeliminate foreign particles and pathogens.

Amongst the diverse array of the humoral immune responses(e.g. clotting cascade, anti-oxidant defense enzymes like super-oxide dismutase, peroxidase, catalase and nitric oxide synthase,defensive enzymes like lysozyme, acid phosphatase and alkalinephosphatase, reactive oxygen and nitrogen intermediates and

P. Amparyup et al. / Fish & Shellfish Immunology 34 (2013) 990e1001 991

antimicrobial peptides), one of the most effective immune mech-anisms of invertebrates against intruder materials is the cellularmelanotic encapsulation. The complex melanization cascaderequires the combination of circulating hemocytes and severalassociated proteins of the prophenoloxidase (proPO)-activatingsystem, and is one of the more potent humoral constituents inmany invertebrates that is responsible for wound healing andparasite entrapment as well as for microbe killing [7e12].

The proPO-activating system serves an important role as a non-self-recognition system that participates in the innate immuneresponses through accompanying with the cellular responses viahemocyte attraction and inducing phagocytosis, melanization,cytotoxic reactant production, particle encapsulation, and theformation of nodules and capsules [8e11].

Melanin biosynthesis requires multiple steps, both enzymaticand non-enzymatic (spontaneous) reactions. The enzymatic reac-tion is catalyzed by the key enzyme phenoloxidase (PO) and thisstep is considered as the rate-limiting step of melanin formation.During the proPO system activation, the subsequent reactiveintermediates, such as quinone-like substances, reactive oxygen(ROI) or nitrogen (RNI) intermediates, which possess cytotoxicactivity toward microorganisms, are produced and restrain theinvasion of microbial pathogens into the host body cavity and assistin wound healing [9,10,13e15]. However, whilst any reductions inthe activity of the proPO system might lead to the failure ofphagocytosis, it is obvious that excess or prolonged levels (i.e.inappropriate levels) of these cytotoxic substances can also lead to

Fig. 1. Schematic outline of the principle components in the prophenoloxidase (proPO)-actiPAMPs (LPS, PGN and b-1,3-glucan) are recognized by the appropriate pattern-recognitionprotein (LGBP) and b-1,3-glucan-binding protein (bGBP)). This event triggers the activation ca(clip-SP) designated as a proPO-activating enzyme (PPAE). Subsequently, the inactive proPOwhich can cross-link neighboring molecules to form melanin around invading microorganisthe common serine proteinase that leads to the production of antimicrobial peptides (AMP

host tissue damage and cell death [10]. Accordingly, it is notsurprising that the activation of the melanization reaction is strictlyregulated and takes place precisely at the site of injury or infection.

Activation of the proPO system is controlled by a multisteppathway. The recognition of microbial PAMPs (elicitors) by PRPsleads to the activation of a series (cascade) of serine proteinases(SPs) and eventually culminates in the proteolytic cleavage of theproPO zymogen to the active PO enzyme. The proPO cascade istriggered upon recognition of the microbial-derived PAMPs,including lipopolysaccharide (LPS) from Gram-negative bacteria,peptidoglycan (PGN) from Gram-positive bacteria and b-1,3-glucans from fungi. Activation of the SP cascade by the PAMPs-PRP interactions, with the final activation of PO, results in theproduction of the polymeric melanin and its deposition precisely atthe site of infections or around the surface of foreign microorgan-isms (Fig. 1) [8].

Over the past decade, the proPO system has been fairly wellstudied and as a result its importance in invertebrate immunesystem has been confirmed. The biochemical pathway and severalassociated proteins involved in the proPO system activation arenow relatively well characterized in many insects and to someextent in crustacean species [10e12]. In some insects, the over-lapping between proPO system and the Toll signaling pathway hasalso been demonstrated [16e20]. This review summarizes thecurrent understanding of the molecular mechanisms and theregulation of the proPO system in penaeid shrimp (Decapoda:Penaeidae), emphasizing the important roles and discussing the

vating system in arthropods. During microbial infection, non-self molecules that act asproteins (PRPs) (peptidoglycan-binding protein (PGBP), LPS and b-1,3-glucan-bindingscade of several serine proteinase (SPs), leading to a final clip-domain serine proteinasezymogen is converted to active phenoloxidase (PO), by PPAE to produce the quinones,ms. The connection between the proPO system and the Toll signaling pathway throughs) is based on the works on the insects T. molitor [16e18] and M. sexta [19,20].

P. Amparyup et al. / Fish & Shellfish Immunology 34 (2013) 990e1001992

current knowledge on the shrimp proPO system in response to thepathogenic microorganisms, including differences compared to thebetter known systems in insects.

2. Pattern-recognition proteins (PRPs) in shrimp proPOsystem

Recognition of PAMPs, such as PGN, LPS and b-1,3-glucans byPRPs is an essential step for the activation of the proPO cascade.Several groups of invertebrate PRPs that bind to PAMPs and activatethe proPO system have been reported [3,10,21e23], and theseinclude the peptidoglycan-recognition proteins (PGRPs) [21,24e28], Gram-negative-binding proteins (GNBPs) [10,23,29], b-glucan-binding proteins (bGBPs) [30e33], LPS and b-1,3-glucan-binding proteins (LGBPs) [29,34,35] and C-type lectins [36,37]. Inshrimp, little is known about the PRPs involved in the proPOcascade. Nevertheless, a few PRPs in shrimp that are involved in theactivation of the proPO system or melanization have been reported[38e40], and are outlined below (Table 1).

2.1. b-Glucan-binding proteins (bGBPs and bGBP-HDLs)

The first group of bGBPs in crustaceans was characterized in thecrayfish, Pacifastacus leniusculus (Decapoda: Astacoidea) [41]. Thecrayfish bGBP transcript is synthesized in the hepatopancreas wherethe encoded protein is released into the plasma and, after binding to

Table 1Genes in proPO system of penaeid shrimp.

Gene name Scientificname

Accessionnumber

Full-lengcDNA (bp

Pattern-recognition proteins (PRPs)bGBP-HDL L. vannamei AY249858 6379

F. chinensis GU461662 6443LGBP P. monodon JN415536 1140

AF368168 1297L. vannamei AY723297 1203F. chinensis AY871267 1277

DQ091256 1253L. stylirostris AF473579 1352M. japonicus AB162766 1098

EU267001 1293C-type lectin-containing domain protein L. vannamei JF834160 1353

Clip-domain serine proteinases (Clip-SPs)PPAE1 P. monodon FJ595215 1529

L. vannamei 1557PPAE2 P. monodon FJ620685 1578

Clip-domain serine proteinase homologs (Clip-SPHs)MasSPH1 P. monodon DQ455050 1958

DQ916148 1530DQ403191 1949

MasSPH2 P. monodon FJ620686 1672EF128030 1740

Prophenoloxidases (proPOs)proPO1 P. monodon AF099741 3002

AF521948 2061F. chinensis EU015060 2061

AB374531 3023P. semisulcatus AF521949 2055L. vannamei EU284136 2471

EF115296 3232AY723296 2061

M. japonicus AB065371 2046AB073223 3047

proPO2 P. monodon FJ025814 2536EU853256 2531

L. vannamei EU373096 2504EF565469 2514

F. chinensis FJ594415 2312proPOb M. japonicus AB617654 2453

b-1,3-glucan, enhances the activation of the proPO system andinduces hemocyte degranulation and opsonization [30,41e44]. Agroup of similar proteins (named bGBP-HDL) to the crayfish bGBPwere isolated from the shrimp Litopenaeus vannamei and Fenner-openaeus chinensis [45e49], and have an approximately 70% aminoacid sequence similarity to the crayfish bGBP. The bGBP/bGBP-HDLfamily contains two glucanase-like motifs that have a high sequencesimilarity to the catalytic regions of microbial b-glucanases, but theylack glucanase activity and have the integrin-recognitionmotif (RGDor RGE) as a putative cell adhesion site. Members of the bGBP/bGBP-HDL family have been isolated and cloned from four penaeid shrimpspecies; Penaeus californiensis [38,45], Litopenaeus stylirostris [46],L. vannamei [45e48] and F. chinensis [49].

The bGBP-HDL of L. vannamei is expressed in the hepatopan-creas, muscle, pleopods and gills, but not in the hemocytes [48].Likewise, the bGBP-HDL of F. chinensis is expressed in the intes-tine, hepatopancreas, muscle and gill tissues, but it is alsoexpressed in the hemocytes, and its expression profile is modifiedafter white spot syndrome virus (WSSV) infection [49]. Althoughthe mRNA transcripts of shrimp bGBP-HDL have been detected inseveral shrimp tissues, as mentioned above, the bGBP-HDLprotein was only found to be present in the plasma [45,46]. InP. californiensis, purified bGBP protein can trigger PO activity inthe presence of b-1,3-glucan and so this bGBP-HDL may bea shrimp PRP for the recognition of b-1,3-glucans in the proPOactivation pathway [38].

th)

Codedamino acid

Signalpeptide

Mature protein(amino acids/MW)

Reference source

1454 e 1454 aa/164.0 kDa [48]2021 e 2021 aa/227.5 kDa [49]366 17 349 aa/39.9 kDa [40]366 17 349 aa/39.9 kDa [55]367 17 350 aa/39.9 kDa [102]366 17 349 aa/39.9 kDa Liu et al. (unpublished)366 17 349 aa/39.8 kDa Du et al. (unpublished)376 27 349 aa/39.9 kDa [50]366 17 349 aa/39.8 kDa Aoki et al. (unpublished)354 23 331 aa/40.2 kDa [54]311 16 295 aa/32.8 kDa [39]

463 18 445 aa/48.7 kDa [75]462 18 444 aa/48.5 kDa [77]371 25 346 aa/36.5 kDa [76]

523 19 504 aa/51.6 kDa [84]509 19 490 aa/50.5 kDa [86]516 19 497 aa/51.0 kDa [86]387 20 367 aa/39.4 kDa [75]386 19 367 aa/39.4 kDa Sriphaijit and Senapin

(unpublished)

688 e 688 aa/78.7 kDa [97]686 e 686 aa/78.5 kDa Ye at al. (unpublished)686 e 686 aa/78.2 kDa [105]686 e 686 aa/78.1 kDa Bae et al. (unpublished)684 e 684 aa/78.0 kDa Ye et al. (unpublished)686 e 686 aa/78.1 kDa Lai et al. (unpublished)686 e 686 aa/78.1 kDa [103]686 e 686 aa/78.1 kDa [102]681 e 681 aa/78.1 kDa [99]688 e 688 aa/80.0 kDa Rojtinnakorn et al. (unpublished)689 e 689 aa/79.2 kDa [98]690 e 690 aa/79.4 kDa Ma and Chan (unpublished)691 e 691 aa/78.8 kDa [101]691 e 691 aa/78.8 kDa [104]687 e 687 aa/78.8 kDa Sun et al. (unpublished)708 18 690 aa/79.1 kDa [100]

P. Amparyup et al. / Fish & Shellfish Immunology 34 (2013) 990e1001 993

2.2. LPS and b-1,3-glucan-binding proteins (LGBPs)

LGBPs, initially isolated from the freshwater crayfish,P. leniusculus, show a significant homology with the insect GNBPs.In the crayfish, the binding of LGBP to LPS or b-1,3-glucan has beendocumented to activate the proPO system [35]. In penaeid shrimp,several LGBPs have been cloned and characterized, such as inP. stylirostris [50], L. vannamei [51], F. chinensis [52,53], Marsupe-naeus japonicus [54] and Penaeus monodon [40,55]. Most of thecharacterized LGBP mature proteins have an average molecularmass of 39.8e40.2 kDa and contain a conserved domain of theglycoside hydrolase family 16 that is comprised of a poly-saccharide-binding, glucanase- and b-glucan-recognition motifsand two RGD motifs [40,54]. LGBP mRNA is mainly expressed inshrimp hemocytes and it appears to be an immune-responsivegene since its transcript expression levels are up-regulated in theearly-mid infection phase response to WSSV or Gram-negativebacteria or their associated LPS [40,50,51,53,54] and down-regulated in response to late infection with Vibrio anguillarum[52]. However, in P. monodon the only LGBP found so far (PmLGBP)acts as a PRP of the proPO cascade [40].

2.3. C-type lectins

C-type lectins are carbohydrate-binding proteins that playessential roles in various biological processes across awide range ofvertebrates and invertebrates [56]. A link between lectin and theproPO system was first reported in the blattarid insect (cockroach)Blaberus discoidalis [57], where purified serum lectins were foundto activate proPO in the cockroach hemolymph and to enhance thelaminarin-stimulated proPO system activation. In the lepidopteraninsect Manduca sexta (tobacco hornworm), two C-type lectinsnamed immulectins-1 (IML-1) and IML-2 have been characterizedas PRPs that are required for the proPO activation in the insect’shemolymph [58,59].

In crustaceans, the connection between C-type lectins andactivation of the proPO system has recently been demonstrated incrayfish [60,61]. The C-type lectin PcLec2 from the red swampcrayfish, Procambarus clarkii, was characterized as a PRP thatparticipated in the proPO activation [60], whilst in the freshwatercrayfish, P. leniusculus, the C-type lectin (mannose-binding lectin;Pl-MBL), which was found to be synthesized in granular cells andexocytosed upon infection, functions as a regulator of the proPOsystem under high Ca2þ concentration in the crayfish hemocytelysate supernatant [61]. In penaeid shrimp, several C-type lectinshave been identified and biochemically characterized (reviewed in[62]), but the potential functions of C-type lectins in activation ofthe shrimp proPO system remain poorly understood. Nevertheless,a connection between C-type lectins and proPO was recently re-ported for the C-type lectin from the shrimp L. vannamei (LvCTLD)that was found to enhance the PO activity in the shrimp’s hemo-lymph [39]. However, the molecular mechanism of shrimp proPOactivation remains to be further elucidated.

3. Clip-domain serine proteinase cascade and the proPO-activating enzyme (PPAE) in the shrimp proPO activation

The activation of the proPO cascade requires the proteolyticsteps of SPs. SPs constitute one of the families of proteolyticenzymes and are involved in mediating several physiologicalprocesses by the proteolytic cleavage of specific proteins. SPs arecharacterized by the presence of three catalytic residues (H, D andS) that form a catalytic triad. Clip-domain SPs (Clip-SPs) belong tothe SP family and have been implicated in the proPO activationcascade [63].

All Clip-SPs are synthesized as inactive zymogens consisting ofan N-terminal clip-domain and a C-terminal catalytic SP domain.The characteristic of clip-domain, usually composed of between 37and 55 amino acid residues, is interlinked by three strictlyconserved disulfide bonds and is connected to the SP domain bya disulfide linkage after proteolytic activation [63]. Clip-SPs can beclassified into catalytic SPs and non-catalytic SPs. The catalytic Clip-SPs, a group of proteolytic enzymes, and the non-catalytic SPs,referred to as Clip-SP homologs (Clip-SPHs), are similar in aminoacid sequence but the serine residue in the active catalytic triads ofClip-SPHs is replaced by glycine. Clip-SPHs do not exhibit anyproteolytic activity and are involved instead in protein interactions[63,64].

The terminal Clip-SPs of the proteolytic cascades of the proPOsystem that converts the proPOs to POs is termed the proPO-activating enzyme (PPAE) [63e77]. PPAEs have been character-ized from several insects and crustaceans, including, within insects,Bombyx mori [65],M. sexta [63,66e69], Holotrichia diomphalia [70e72] and Tenebrio molitor [73], and, within crustaceans, the crayfishP. leniusculus [74] and the shrimp P. monodon [75,76] andL. vannamei [77].

The penaeid shrimp PPAE was first cloned from P. monodon andnamed PmPPAE1 [75]. Subsequently, a second PPAE (PmPPAE2) [76]and several clip-SPs have been identified and cloned from a varietyof shrimp species, including P. monodon [78], F. chinensis [79],Fenneropenaeus indicus [80] and L. vannamei [77]. However, so faronly three of these clip-SPs, two from P. monodon (PmPPAE1 andPmPPAE2) [75,76] and one from L. vannamei (LvPPAE1) [77], havebeen reported to be involved in the proPO system (Table 1).

Both PmPPAE transcripts (PmPPAE1 and PmPPAE2) are mainlyexpressed in shrimp hemocytes [75,76]. PmPPAE1 appears to be animmune-responsive gene since its transcript expression level is up-regulated after Vibrio harveyi infection. Immunoblotting analysis ofthe hemocyte and plasma using a polyclonal antibody against theSP domain of PmPPAE1 indicates that the protein is localized inhemocytes [75]. In the shrimp L. vannamei, LvPPAE1 mRNA levelswere down-regulated in hemocytes and up-regulated in the gillafter challenge with V. harveyi [77]. In contrast, in the crayfishP. leniusculus, the PPAE1 transcript levels were not affectedfollowing Aeromonas hydrophila infection [81]. In insects, theM. sexta PPAE (PAP-1) is constitutively expressed at high levels inthe integument and less abundantly in the fat body of naive larvae.However, the transcript expression levels of PAP-1 and the twoother PPAE transcripts (PAP-2 and PAP-3) are up-regulatedfollowing bacterial infection [66e68]. The differences in theexpression levels of PPAEs among species can be explained as beingdue to the differences in the pathogenicity or virulence of themicroorganisms in each species.

Arthropod clip-SPHs have been shown to be involved in variousbiological functions including immunity [63]. Clip-SPH proteins,which lack SP enzymatic activity, are regulators in the proPOsystem. In insects, the proPO-activating factor (PPAF)-II of thebeetle, H. diomphalia, is a clip-SPH and functions as a proteincofactor for PPAF-I [82], whilst SPH1 and SPH2 of the lepidopteranM. sexta (tobacco hornworm) serve as protein cofactors for the twoPPAEs (PAP-2 and PAP-3) in the proPO activation [66e68]. Incrustaceans, two SPHs (PlSPH1 and PlSPH2) from the crayfishP. leniusculus have been reported to be involved in the PGN-inducedproPO activation and probably function as protein cofactors in thePGN-binding complex with LGBP [83].

In the shrimp P. monodon, the two known clip-SPHs(PmMasSPH1 and PmMasSPH2) have a high amino acid sequencesimilarity (74% and 69%) to the crayfish PlSPH2 and PlSPH1,respectively ([84], Amparyup et al., unpublished data). PmMasSPH1is a multifunctional immune molecule that contains extensive

P. Amparyup et al. / Fish & Shellfish Immunology 34 (2013) 990e1001994

glycine-rich repeats (LGQGGG) and a clip-domain at the N-terminus and C-terminal SP domain. PmMasSPH1 transcript levelsare induced in response to infection with the shrimp pathogenicbacterium V. harveyi [84]. The N-terminal region of PmMasSPH1exhibits in vitro antibacterial activity against Gram-positivebacteria; the SP-like domain mediates the hemocyte adhesionand displays bacterial-binding activity to V. harveyi and the LPS ofEscherichia coli [85]. In accord, PmMasSPH1 displays an in vivoopsonic activity against V. harveyi. In addition, P. monodon SPH516,an isoform of PmMasSPH1, specifically interacts with the metal ion-binding (MIB) domain of the yellow head virus (YHV), suggestingthat it might play an important role in viral responses [86]. More-over, dsRNA interference studies revealed significant decreases inthe hemolymph PO activity of PmMasSPH1 and PmMasSPH2knockdown shrimp, suggesting that both SPHs are involved in theshrimp proPO system (Amparyup et al., unpublished data). In twoother shrimp (M. japonicus and F. chinensis), at least three clip-SPHsgenes have been identified [79,87,88], yet none of these clip-SPHshave so far been implicated in the proPO system. However, datafrom molecular studies have revealed significant changes in thegene expression levels of these clip-SPH transcripts followingmicrobial infection, suggesting that theymay be involved in shrimpimmunity and in the proPO system upon infection. Regardless,further studies are still required to elucidate the function of theseproteins in the proPO system.

4. Prophenoloxidase (proPO): a key enzyme in the proPOsystem

PO, a crucial component of the arthropod proPO system and thekey enzyme in the synthesis of melanin, is a bifunctional coppercontaining enzyme that belongs to the type-3 copper protein familyand possesses both tyrosinase/monophenolase (EC 1.14.18.1) andcatecholase/diphenoloase (EC 1.10.3.1) activities that in turnconvert a variety of monophenols and o-diphenols into o-quinonethat ultimately act as precursors for melanin production [8,9].Biochemical investigation in insects and some crustaceans indi-cated that PO is generally synthesized and maintained as theenzymatically inactive precursor proPO, with an approximatemolecular mass of about 70e80 kDa, and is coordinated with twoatoms of copper [7,11]. Activation of proPO is triggered bymicrobialelicitors (PAMPs), including LPS, PGN and b-1,3-glucan, and resultsin the limited proteolytic cleavage at the N-terminal by the specificPPAE proteinases [7,8,11]. Significantly, POs also share a homolo-gous sequencewith the oxygen carrier proteins (hemocyanins), andso it is not surprising that some PO-like activity was detected fromhemocyanin under certain conditions [89e94].

The sequence of arthropod proPO gene was firstly identifiedfrom the freshwater crayfish P. leniusculus [95]. Subsequently,various arthropod proPO sequences have been reported from anarray of insect and crustacean species. In insects, the number of theproPO genes found in each species varies widely, ranging from tenand nine in the mosquitoes Aedes aegypti and Anopheles gambiae,respectively, to three in the fruit fly Drosophila melanogaster, two inthe beetle H. diomphalia, the silkworm B. mori and the tobaccohornworm M. sexta, down to only a single proPO gene in thehoneybee Apis mellifera (reviewed in [10e12]).

In shrimp, biochemical investigations of various aspects ofproPO have been performed so far in six different shrimp species[96e105]. The first penaeid proPO was successfully purified fromthe hemocytes of P. californiensis in 1999 [96]. Thereafter, a numberof proPO genes have been identified and cloned from variousshrimp species, including one proPO gene in Penaeus semisulcatus(Genbank accession no: AF521949), two proPO genes in each of

P. monodon [97,98], M. japonicus [99,100], L. vannamei [101e104]and F. chinensis [105] (Table 1).

In the insect species for which good genomic or other suitablegenome coverage data are available, the presence of different proPOgenes have been reported, as exemplified above. However, it hasbeen suggested the proPO genes of the malaria vector A. gambiaeare differentially expressed in different developmental stages,whilst some of them are induced after blood feeding [106e108].Note that after blood feeding the insect is potentially exposed tohost animal-blood borne pathogens. In shrimp, the expressionstudies of the proPO genes during development was first done inP. monodon with an extremely low level at the N4 stage and theexpression was gradually increased in the later stages of develop-ment [109]. The expression level of proPO genes was furtherconfirmed and extended to include PmproPO1 and PmproPO2 andfound to be differentially expressed in different stages of larvaldevelopment with PmproPO2 and PmproPO1 potentially being themajor proPO gene in the early and late developmental stages,respectively [76].

Shrimp proPOs have been shown to be localized only in thehemocytes [101,104]. Accordingly, all the penaeid shrimp proPOmRNA transcripts, except for one from M. japonicus [100], are re-ported to be expressed exclusively in hemocytes [97,98,105]. This isin accord with previous studies in other crustaceans which havedemonstrated that the enzymes of the proPO system are synthe-sized in hemocytes, localized in the granules of semigranular andgranular hemocytes and released into the hemolymph by degran-ulation upon infection (reviewed in [10]). Nonetheless, a proPO(proPOb) gene identified from the plasma of the kuruma shrimpM. japonicus has recently been reported to bemainly synthesized inhepatopancreas [100]. Unlike other arthropod proPOs, proPObcontained a signal peptide-like sequence at its N-terminus anddisplayed a PO activity in shrimp plasma and these physicochem-ical properties are resemble to those of arthropod hemocyanins,which are synthesized in hepatopancreas and then released toplasma, have a typical signal peptide and exhibit the plasma POactivity upon wounding or infections [91e94,100]. However, thisproPOb protein is supposed to be a crustacean proPO (but nota hemocyanin) due to its primary structure as well as its enzymaticproperties are muchmore similar to those of the crustacean proPOsrather than other hemocyanins [100].

All currently known shrimp proPOs exhibit the typical charac-teristic of arthropod proPOs, including having two functionalcopper-binding sites (copper-binding sites A and B), a proteolyticactivation site, and no hydrophobic signal peptide. As with othermembers of the type-3 copper protein family, shrimp proPOscontain a canonical type-3 bi-nuclear active site with six structur-ally conserved histidine residues within the two annotated copper-binding sites (A and B). Almost all currently available sequences ofshrimp proPOs have been shown to belong to the penaeid shrimpproPO clade of the crustacean proPOs group, which contains thetwo sub-distinct groups of proPO1 and proPO2 [98]. The exception,however, is the novel proPO-like protein (proPOb) identified fromM. japonicus hemolymph, which differed from the previously re-ported crustacean’s proPO due to their cDNA and deduced aminoacid sequences showed low sequence identity (approximately 30%)with the previously reported proPO sequences (proPO1 andproPO2) of penaeid shrimp [100].

5. Negative regulation of the proPO cascade

Melanization, induced by activation of the proPO system, isessential for the immune defense. However, the melanin reaction istightly controlled at multiple levels, including by inhibition of theSP cascade by proteinase inhibitors, the PO activity by PO inhibitors,

P. Amparyup et al. / Fish & Shellfish Immunology 34 (2013) 990e1001 995

and the melanin reaction products by melanization-inhibitingprotein (MIP). This tight control is required since excess melaninreaction products (highly reactive and toxic quinone intermediates)can cause damage and death to the host cells. Unfortunately,studies on the inhibitors that negatively regulate the proPO systemor melanization in penaeid shrimp are not yet available. Rather,only the nucleotide sequence and transcript expression profiles ofserpins [110,111] and MIP [112] homologs have been reported todate.

Various proteinase inhibitors, including members of the serpinand pacifastin superfamily, have been functionally characterized asnegative regulators of the proPO activation cascade by inhibitingthe SPs in different insect and crustacean species [10]. Serpin isa class of proteinase inhibitors that act as suicide-like substrates bybinding covalently to their specific target proteinases. In insects,serpins from D. melanogaster (Serpin-5, -27A, -28D and -77Ba)[113e117], A. gambiae (SRPN2) [118,119], M. sexta (Serpin-1J, -3, -4,-5 and -6) [22,68,120e122] and T. molitor (SPN48) [123] have beenidentified as negative regulators of the melanization cascade.However, in crustaceans, so far only one SP inhibitor, pacifastin,from the crayfish P. leniusculus has been reported to be a specificinhibitor for PlPPAE1. This crayfish pacifastin has a molecular massof 155 kDa and contains a light chain with nine likely proteinaseinhibitor domains of the pacifastin family and a heavy chain con-taining three transferrin lobes [124]. In shrimp, only a few serpinshave been reported, but their expression profiles suggest that theyare likely to be involved in the shrimp immune response againstbacterial and viral infections [110,111]. However, the function ofthese genes in the proPO system has not been investigated.

MIP was originally identified in the coleopteran insect T. molitor,and was found to decrease the melanization reaction by inhibitingthe melanin synthesis from quinone compounds, but not to affectthe PO activity [125]. In crustaceans, the MIP homolog from thecrayfish, P. leniusculus (PlMIP) has an amino acid sequence simi-larity (approximately 55%) to ficolin, a lectin protein and shares anamino acid sequence similarity with the aspartic acid rich motif ofthe T. molitor MIP. The PlMIP functions in preventing the melanin-forming reactions from quinone and also the PPAE activity [126].In shrimp, a homolog of MIP has been identified in P. monodonwithan 80% amino acid sequence similarity to the crayfish PlMIP [112].However, the function of the shrimp PmMIP has not been experi-mentally verified.

In the absence of any information on these inhibitors, the under-standing of the molecular mechanisms by which inhibitors controlthe melanization cascades in penaeid shrimp remains incomplete.

6. POs are important in shrimp immunity

The importance of the proPO activation system/melanization inthe host’s defense against pathogen infections in shrimp and othercrustaceans has been investigated recently using in vivo genesilencing. Although in some insects it has been reported that theproPO system is not essential for defense against microbial infec-tions [127,128], in crayfish the suppression of the proPO gene usingRNAi-mediated gene knockdown of the P. leniusculus proPO genesuggested a potentially important role of proPO in crayfish immu-nity [81]. Likewise, in penaeid shrimp the proPO systemwas foundto be essential for the shrimp’s defense against pathogenic micro-organisms as follows. In F. chinensis, proPO transcripts were up-regulated in response to V. anguillarum infection [105], whilst inL. vannamei, the expression levels of proPO1 and proPO2 were bothdown-regulated after WSSV infection [103,104] or followingV. alginolyticus challenge as well as after ecdysone treatment [129].In P. monodon, in which the significant role of proPOs was firstclarified, dsRNA-mediated gene knockdown of the proPO system

component (two proPO and two PPAE transcripts) significantlyreduced the total PO activity in the shrimp hemolymph as well asenhancing the susceptibility of the shrimp to infection witha pathogenic isolate of V. harveyi [75,76,98]. InM. japonicus, dsRNA-mediated gene knockdown of the proPO gene resulted in a reduc-tion in the hemocyte count, an increase in the bacterial load in thehemolymph even in the absence of a bacterial or viral challenge,and in an acute increase in the shrimp mortality [130]. InL. vannamei, PPAE gene (as LvPPAE1) knockdown reduces thesurvival of shrimp after V. harveyi infection, supporting an impor-tant role for shrimp PPAE in the defense against bacterial invasion[77]. Overall, these in vivomolecular investigations suggest that theproPO system is critical for the defense against pathogenic infec-tions, especially in both P. monodon and M. japonicus, as well as inpenaeid shrimp in general [75,76,98,130].

Significantly, silencing of proPO genes through dsRNA interfer-ence affected the expression levels of other immune gene tran-scripts in both penaeid shrimp examined [40,130]. Down-regulation of the M. japonicus proPO gene led to a reducedexpression level of the antimicrobial peptide (AMP) genes,including lysozyme, crustin and penaeidin [130], whereas down-regulation of the two proPO dsRNA of P. monodon significantlydecreased the transcription levels of two genes in the proPO system(PmLGBP and PmPPAE2), and two other AMP genes (PEN3 and Crus-likePm) [40]. Obviously, the proPO system is somehow interrelatedwith other branches of the crustacean immune system, and thereare several reports in the literature on insect immunity that suggesta correlation between the activation of the proPO system and theAMP synthesis. The molecular activation mechanism demonstratedthe overlapping between the proPO system and the Toll signalingpathway has been firstly reported in the insect T. molitor [16e18].The Lysine-type PGN and b-1,3-glucan-dependent proPO systemand Toll signaling cascade shared three activating SPs: modularserine protease (MSP), Spätzle-processing enzyme-activatingenzyme (SAE) and Spätzle-processing enzyme (SPE), in which theSPE has been identified as a terminal proteinase that cleaves bothproPO precursor and pro-Spätzle that lead to the respectiveproduction of melanin and AMPs [16e18]. The molecular cross-talkbetween Toll and proPO cascade has been then confirmed in theinsect M. sexta in which the hemolymph proteinase-6 (HP6)participates in the proPO activation by processing of the proPAP-1and also takes part in the Toll pathway through the proteolyticcleavage of the upstream hemolymph proteinase-8 (HP8) thatcleaves and activates the pro-Spätzle to initiate the expression ofAMPs [19,20]. However, this important insight into the regulationof the proPO system and the molecular cross-talk between AMPproduction and the proPO system has not been evaluated let aloneconfirmed in shrimp immunity and so awaits further studies.

7. The proPO system in P. monodon

The shrimp proPO system has been investigated for more than12 years. However, the immune cascade leading to activation ofinactive proPO to active PO in arthropods is still largely undescribedin penaeid shrimp, with most available data being available insteadfrom insect species. In this part, we review the research on theproPO system of the shrimp P. monodon. Using EST databasesearching (the complete annotated genome sequence of a penaeidshrimp is not yet available) and PCR-based approaches, 10 full-length cDNAs of two proPOs, four SPs, three SPHs and one PRPhave been identified in P. monodon. Themolecular characteristics ofthese genes are shown in Table 2 and Fig. 2. RNAi-mediated genesilencing has been used to test the function of eight of thesecandidate genes in the proPO system in P. monodon, fromwhich theknockdown in expression levels of seven of these genes (PmproPO1,

Table 2Molecular characteristics of the genes and their predicted protein products in the proPO system of the penaeid shrimp P. monodon.

Gene name CDS/ORF Closest gene(% amino acidsimilarity)

Putative N-glycosylationsites

Conserved domains Biological functions/reference sources

Pattern-recognition protein (PRP)PmLGBP 1101 bp/

366 aaP. monodon bGBP (99%) NRS(66) and NLS(318) Polysaccharide-binding motif,

glucanase- andb-glucan-recognition motifs andintegrin-recognition motifs (RGD)

Pattern-recognition protein for LPS andb-1,3-glucan in theshrimp proPO system [40]

Clip-domain serine proteinases (Clip-SPs)PmPPAE1 1392 bp/

463 aaP. leniusculus PPAE (70%) NGS(42) and NAT(192) Clip-domain and serine proteinase

domainPPAE in the shrimp proPO system thatpossibly mediates theactivation of PmproPO2 and importantin shrimp immunity [75]

PmPPAE2 1116 bp/371 aa

M. sexta PAP-1 (51%) NVT(84) Clip-domain and serine proteinasedomain

PPAE in the shrimp proPO system thatpossibly mediates theactivation of PmproPO1 and importantin shrimp immunity [76]

PmClipSP1 1101 bp/366 aa

A. gambiae Serine protease14D (57%)

NFS(219) and NKS(228) Clip-domain and serine proteinasedomain

Not required for shrimp proPO cascadebut still plays a potentialrole in the antibacterial defensemechanism in shrimp immuneresponse [78]

PmClipSP2 1107 bp/369 aa

D. melanogaster melanizationprotease-1 (52%)

NPT(25), NTS(156), NGS(159)and NRT (184)

Clip-domain and serine proteinasedomain

Not determined (Amparyup et al.,unpublished data)

Clip-domain serine proteinase homologs (Clip-SPHs)PmMasSPH1 1572 bp/

523 aaP. leniusculus SP-like 2a(74%)

NDT(28) and NDT (203) Clip-domain and serineproteinase-like domain

Multifunctional immune moleculeand function in the shrimpproPO system [84,86] (Amparyup et al.,unpublished data)

PmMasSPH2 1164 bp/387 aa

P. leniusculus SP-likeprotein-1 (68%)

Not found Clip-domain and serineproteinase-like domain

Function in shrimp proPO system(Amparyup et al.,unpublished data)

PmMasSPH3 1164 bp/387 aa

P. monodon MasSPH1 (57%) NTT(12), NET(85), NCS(113),NTT(126) and NVT(246)

Clip-domain and serineproteinase-like domain

Not determined (Amparyup et al.,unpublished data)

Prophenoloxidases (proPOs)PmproPO1 2067 bp/

688 aaP. monodon proPO1 (100%) NET(117), NQT(170), NTS(571)

and NTT(662)Copper-binding sites A and B Major component of proPO system and

important in shrimp immunity [98]PmproPO2 2070 bp/

689 aaP. monodon proPO1 (81%) NET(119), NET(172), NNS(275),

NIS(357) and NLS(432)Copper-binding sites A and B Major component of proPO system and

important in shrimp immunity [98]

Fig. 2. Schematic illustration of the primary structure of the genes in the proPO system of the penaeid shrimp P. monodon. LGBP: PmLGBP (Genbank accession no. JN415536). PPAEs:PmPPAE1 (FJ595215) and PmPPAE2 (FJ620685). proPOs: PmproPO1 (AF099741) and PmproPO2 (FJ025814).

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P. Amparyup et al. / Fish & Shellfish Immunology 34 (2013) 990e1001 997

PmproPO2, PmPPAE1, PmPPAE2, PmMasSPH1, PmMasSPH2 andPmLGBP) significantly decreased the hemolymph PO activity, sup-porting that these proteins function in the proPO system of thisshrimp ([40,75,76,98], Amparyup et al., unpublished data). Incontrast, the knockdown of PmClipSP1, which has a 57% amino acidsequence similarity to the A. gambiae serine protease 14D, did notalter the hemolymph PO activity, but resulted in an increasednumber of bacteria in the hemolymph and in the mortality rate ofshrimp infected with V. harveyi [78]. As to the remaining twouncharacterized proteins (PmClipSP2 and PmMasSPH3), based onanalysis of their amino acid sequences for homology, they mayfunction in the proPO cascade. These are discussed with respect totheir functional categories below.

7.1. The PRP of P. monodon

The P. monodon LGBP (PmLGBP) was recently reported to act asa PRP, binding to LPS or b-1,3-glucan and activating the proPOsystem [40]. This protein was initially identified as a bGBP inP. monodon, based on its binding activity to b-1,3-glucan but not toLPS [55]. However, recent investigations have identified a PmLGBPthat binds bacterial LPS and b-1,3-glucan with a dissociationconstant of 6.86 � 10�7 M and 3.55 � 10�7 M, respectively, andactivates the proPO system in the presence of LPS or b-1,3-glucan[40]. Gene silencing of PmLGBP resulted in a significant decreasein the total PO activity, but no significant effect on the transcript

Fig. 3. A proposed mechanism for the activation of the proPO cascade in the penaeid shrimpprophenoloxidase activating enzymes (PPAEs); PmMasSPH1 and PmMasSPH2 are clip-domenoloxidases (proPOs).

expression levels of genes in the proPO system (PmproPO1,PmproPO2, PmPPAE1 and PmPPAE2), AMPs (PEN3, Crus-likePm,SWDPm2 and lysozyme) or the Toll receptor. These results suggestthat the expression of shrimp AMPs is independent of PmLGBPsignaling pathway [40].

7.2. PPAEs of P. monodon

The role of PPAE in the proPO or melanization cascade is welldocumented in insects. Within penaeid shrimp and P. monodon,PmPPAE1 and PmPPAE2 were the first candidate genes to be testedfor their role in the proPO system. PmPPAE1 is more closely relatedto the crustacean PPAE (a crayfish PPAE) [74], and is comprised ofa long glycine-rich region, a clip-domain, a short proline-rich regionat the N-terminus and a C-terminal SP domain [75]. In contrast,PmPPAE2 is very similar to that of insect PPAEs and contains only anN-terminal clip-domain and a C-terminal SP domain [76]. Thecharacterization of their genomic organization also supports thatPmPPAE1 and PmPPAE2 are structurally different. The PmPPAE1gene displays a ten-exon/nine-intron structure, whereas PmPPAE2consists of eight exons and seven introns, supporting that the twoPmPPAEs are encoded from different genes and that they do notarise as a result of alternative exon splicing [76].

PPAE and SPH have been found to be important for the proPOsystem in shrimp. Gene knockdown of PmPPAE1 and PmPPAE2showed a significant reduction (37% and 41%, respectively) in the

P. monodon. PmLGBP is a pattern-recognition protein (PRP); PmPPAE1 and PmPPAE2 areain serine proteinase homologs (Clip-SPHs) and PmproPO1 and PmproPO2 are proph-

P. Amparyup et al. / Fish & Shellfish Immunology 34 (2013) 990e1001998

PO activity, whereas co-silencing of both PPAEs together resulted inno further significant reduction (41%) in the hemolymph POactivity. This implies that both PmPPAE1 and PmPPAE2 are, at leastin part, members of the proPO activation cascade and play a role inthe same branched-proPO pathway. Moreover, the two SPHs,PmMasSPH1 and PmMasSPH2 were characterized in P. monodon bygene silencing and the results indicate that the hemolymph POactivity of PmMasSPH1 and PmMasSPH2 knockdown shrimp weresignificantly decreased after injection of their correspondeddsRNAs, supporting that both SPHs are likely to be involved in theshrimp proPO system (Amparyup et al., unpublished data).Although it is not yet clear whether these SPHs function as a proteincofactor of PPAEs, we suspect that they may act as cofactors in theproPO cascade.

7.3. ProPOs of P. monodon and their relationship to the PPAEs

The two proPO isoforms so far identified in P. monodon,PmproPO1 and PmproPO2, are mainly expressed in hemocytes.PmproPO1 and PmproPO2 have similar predicted molecular massesof 78.7 kDa and 79.2 kDa, respectively, and do not contain a signalpeptide. PmproPO1 shows an amino acid sequence identity andsimilarity with PmproPO2 of 67% and 81%, respectively. AlthoughPO has long been considered as a key enzyme in the proPO system,direct evidence showing the exact roles of PO in shrimp immunityis not available. Within P. monodon, the suppression of PmproPO1,PmproPO2 or both gene transcript levels by dsRNA-mediated RNAiwas shown to efficiently and specifically reduce the endogenousexpression of their respective transcripts, and to significantlydecrease the PO activity by 75%, 73% and 88%, respectively, whenusing L-dopa as substrates [98]. This in vivo assay clearly shows thatboth PmproPOs are key enzymes contributing to the PO activity andare also essential components of the biochemical pathway requiredfor the proPO system in the P. monodon hemolymph. The meaningof the slight synergy observed in the double-knock down is unre-solved between two overlapping or convergent pathways from thatof just an enhanced efficiency of two parallel (redundant) or tissuespecific components in the same pathway.

The relationship of shrimp PPAE and proPO gene expressions inthe larval developmental stages has been investigated [76].PmPPAE1 and PmproPO2 transcripts were expressed in all larvalstages (nauplius, protozoa, mysis and post-larvae), whereasPmPPAE2 and PmproPO1 transcripts were mainly expressed in thelate larval developmental stages (mysis and post-larvae). Thisdifferential gene expression profile probably suggests thatPmPPAE1 is likely to function as the proteinase activator ofPmproPO2 during the early larval developmental stage, whilstPmPPAE2 might act as the activating proteinase of the PmproPO1 inthe later stages of the larval development [76]. However, confir-mation that PmPPAEs directly cleaves PmproPOs (or not) is yet to beestablished.

7.4. Model of the P. monodon proPO cascade

In this review, we propose a model of the proPO activationcascade in P. monodon, based on the data derived from geneknockdown and biochemical experiments. This model is shownschematically in Fig. 3. Initially, PmLGBP acts a functional PRP forLPS and b-1,3-glucan detection and so plays a role in the recogni-tion of microbes and the activation of the proPO system. This PRP-PAMP complex then probably activates the clip-SP cascades thatcan convert the inactive shrimp PPAEs (PmPPAE1 and PmPPAE2) toactive PPAEs. Stimulation of PmPPAE1 and PmPPAE2 leads to theactivation of PmproPO1 and PmproPO2 (to PO1 and PO2), resultingin the production of melanin and reactive oxygen compounds.

Interestingly, specific down-regulation of these two proPOssignificantly decreased the gene expression levels of the two AMPs(PEN3 and Crus-likePm) and two genes in the proPO system(PmLGBP and PmPPAE2), supporting a likely cross-talk between theproPO system and AMP gene synthesis in P. monodon.

8. Conclusions

Melanization is an important mechanism in the immune systemof shrimp as with other arthropods. In recent years, an increasingnumber of functional-characterized genes (PRPs, Clip-SPs andproPOs) in the proPO system, somewhat comparable to those ininsects or other invertebrate species, have been described frompenaeid shrimp. By binding to specific pathogen molecules(PAMPs), the PRPs (i.e. LGBP, GBP and/or C-type lectin) detectpathogen infections and subsequently activate Clip-SPs, PPAE andproPOs to finally activate PO and generate the melanin and theirreaction products (reactive oxygen compounds) that entrap and/orare toxic to pathogens. This review highlights the rational that theproPO system is an important immune system for fighting againstpathogens in penaeid shrimp. However, there is little research onthe mechanism of the proPO system in shrimp. The study of thegenes and proteins involved in this system should be furtherinvestigated to unveil the mechanism of shrimp proPO system.

Acknowledgments

The work on the proPO system in P. monodonwas supported bygrants from the Thailand Research Fund and Thailand NationalCenter for Genetic Engineering and Biotechnology (BIOTEC) (toP.A.), and the Higher Education Research promotion and NationalResearch University Project of Thailand, Office of the HigherEducation Commission (FW 643A), and the National ResearchUniversity Project of Commission for Higher Education, andthe Ratchadaphiseksomphot Endowment Fund. A postdoctoralfellowship from the Ratchadaphiseksomphot Endowment Fund,Chulalongkorn University, is acknowledged (to W.C.).

References

[1] Tassanakajon A, Amparyup P, Somboonwiwat K, Supungul P. Cationic anti-microbial peptides in penaeid shrimp. Marine Biotechnology (NY) 2010;12:487e505.

[2] Hoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RA. Phylogenetic perspec-tives in innate immunity. Science 1999;284:1313e8.

[3] Janeway Jr CA, Medzhitov R. Innate immune recognition. Annual Review ofImmunology 2002;20:197e216.

[4] Medzhitov R. Recognition of microorganisms and activation of the immuneresponse. Nature 2007;449:819e26.

[5] Ishii KJ, Koyama S, Nakagawa A, Coban C, Akira S. Host innate immunereceptors and beyond: making sense of microbial infections. Cell Host andMicrobe 2008;3:352e63.

[6] Lemaitre B, Hoffmann J. The host defense of Drosophila melanogaster. AnnualReview of Immunology 2007;25:697e743.

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

[8] Cerenius L, Söderhäll K. The prophenoloxidase-activating system in inver-tebrates. Immunological Reviews 2004;198:116e26.

[9] Nappi AJ, Christensen BM. Melanogenesis and associated cytotoxic reactions:Applications to insect innate immunity. Insect Biochemistry and MolecularBiology 2005;35:443e59.

[10] Cerenius L, Lee BL, Söderhäll K. The proPO-system: pros and cons for its rolein invertebrate immunity. Trends in Immunology 2008;29:263e71.

[11] Kanost MR, Gorman MJ. Phenoloxidases in insect immunity. In: Beckage NE,editor. Insect immunology. San Diego: Academic Press Inc; 2008. p. 69e96.

[12] Cerenius L, Kawabata S, Lee BL, Nonaka M, Söderhäll K. Proteolytic cascadesand their involvement in invertebrate immunity. Trends in BiochemicalScience 2010;35:575e83.

[13] Zhao P, Li J, Wang Y, Jiang H. Broad-spectrum antimicrobial activity of thereactive compounds generated in vitro by Manduca sexta phenoloxidase.Insect Biochemistry and Molecular Biology 2007;37:952e9.

P. Amparyup et al. / Fish & Shellfish Immunology 34 (2013) 990e1001 999

[14] Cerenius L, Babu R, Söderhäll K, Jiravanichpaisal P. In vitro effects on bacterialgrowth of phenoloxidase reaction products. Journal of InvertebratePathology 2010;103:21e3.

[15] Zhao P, Lu Z, Strand MR, Jiang H. Antiviral, anti-parasitic, and cytotoxiceffects of 5,6-dihydroxyindole (DHI), a reactive compound generated byphenoloxidase during insect immune response. Insect Biochemistry andMolecular Biology 2011;41:645e52.

[16] Kim CH, Kim SJ, Kan H, Kwon HM, Roh KB, Jiang R, et al. A three-stepproteolytic cascademediates the activation of the peptidoglycan-induced Tollpathway in an insect. Journal of Biological Chemistry 2008;283:7599e607.

[17] Kan H, Kim CH, Kwon HM, Park JW, Roh KB, Lee H, et al. Molecular control ofphenoloxidase-induced melanin synthesis in an insect. Journal of BiologicalChemistry 2008;283:25316e23.

[18] Roh KB, Kim CH, Lee H, Kwon HM, Park JW, Ryu JH, et al. Proteolytic cascadefor the activation of the insect toll pathway induced by the fungal cell wallcomponent. Journal of Biological Chemistry 2009;284:19474e81.

[19] An C, Ishibashi J, Ragan EJ, Jiang H, Kanost MR. Functions of Manduca sextahemolymph proteinases HP6 and HP8 in two innate immune pathways.Journal of Biological Chemistry 2009;284:19716e26.

[20] An C, Jiang H, Kanost MR. Proteolytic activation and function of the cytokineSpätzle in the innate immune response of a lepidopteran insect, Manducasexta. FEBS Journal 2010;277:148e62.

[21] Yu XQ, Zhu YF, Ma C, Fabrick JA, Kanost MR. Pattern recognition proteins inMan-duca sexta plasma. Insect Biochemistry and Molecular Biology 2002;32:1287e93.

[22] Kanost MR, Jiang H, Yu XQ. Innate immune responses of a lepidopteraninsect, Manduca sexta. Immunological Reviews 2004;198:97e105.

[23] Iwanaga S, Lee BL. Recent advances in the innate immunity of invertebrateanimals. Journal of Biochemistry and Molecular Biology 2005;38:128e50.

[24] Yoshida H, Kinoshita K, Ashida M. Purification of a peptidoglycan recognitionprotein from hemolymph of the silkworm, Bombyx mori. Journal of BiologicalChemistry 1996;271:13854e60.

[25] Takehana A, Katsuyama T, Yano T, Oshima Y, Takada H, Aigaki T, et al.Overexpression of a pattern-recognition receptor, peptidoglycan-recognition protein-LE, activates imd/relish-mediated antibacterialdefense and the prophenoloxidase cascade in Drosophila larvae. Proceed-ings of the National Academy of Sciences of the United States of America2002;99:13705e10.

[26] Lee MH, Osaki T, Lee JY, Baek MJ, Zhang R, Park JW, et al. Peptidoglycan recog-nition proteins involved in 1,3-b-D-glucan-dependent prophenoloxidase acti-vation system of insect. Journal of Biological Chemistry 2004;279:3218e27.

[27] Charroux B, Rival T, Narbonne-Reveau K, Royet J. Bacterial detection byDrosophila peptidoglycan recognition proteins. Microbes and Infection 2009;11:631e6.

[28] Sumathipala N, Jiang H. Involvement of Manduca sexta peptidoglycanrecognition protein-1 in the recognition of bacteria and activation ofprophenoloxidase system. Insect Biochemistry and Molecular Biology 2010;40:487e95.

[29] Kim YS, Ryu JH, Han SJ, Choi KH, Nam KB, Jang IH, et al. Gram-negativebacteria-binding protein, a pattern recognition receptor for lipopolysaccha-ride and b-1,3-glucan that mediates the signaling for the induction of innateimmune genes in Drosophila melanogaster cells. Journal of Biological Chem-istry 2000;275:32721e7.

[30] Cerenius L, Liang Z, Duvic B, Keyser P, Hellman U, Palva ET, et al. Structureand biological activity of a 1,3-b-D-glucan-binding protein in crustaceanblood. Journal of Biological Chemistry 1994;269:29462e7.

[31] Ma C, Kanost MR. A b1,3-glucan recognition protein from an insect, Manducasexta, agglutinates microorganisms and activates the phenoloxidase cascade.Journal of Biological Chemistry 2000;275:7505e14.

[32] Jiang H, Ma C, Lu ZQ, Kanost MR. b-1,3-Glucan recognition protein-2 (bGRP-2) from Manduca sexta: an acute-phase protein that binds b-1,3-glucan andlipoteichoic acid to aggregate fungi and bacteria and stimulate proph-enoloxidase activation. Insect Biochemistry and Molecular Biology 2004;34:89e100.

[33] Wang Y, Sumathipala N, Rayaprolu S, Jiang H. Recognition of microbialmolecular patterns and stimulation of prophenoloxidase activation by a b-1,3-glucanase-related protein in Manduca sexta larval plasma. InsectBiochemistry and Molecular Biology 2011;41:322e31.

[34] Beschin A, Bilej M, Hanssens F, Raymakers J, Van Dyck E, Revets H, et al.Identification and cloning of a glucan- and lipopolysaccharide-bindingprotein from Eisenia foetida earthworm involved in the activation ofprophenoloxidase cascade. Journal of Biological Chemistry 1998;273:24948e54.

[35] Lee SY, Wang R, Söderhäll K. A lipopolysaccharide- and b-1,3-glucan-bindingprotein from hemocytes of the freshwater crayfish Pacifastacus leniusculus:purification, characterization, and cDNA cloning. Journal of BiologicalChemistry 2000;275:1337e43.

[36] Yu XQ, Kanost MR. Immulectin-2, a pattern recognition receptor thatstimulates hemocyte encapsulation and melanization in the tobacco horn-worm, Manduca sexta. Developmental and Comparative Immunology 2004;28:891e900.

[37] Yu XQ, Ling E, Tracy ME, Zhu Y. Immulectin-4 from the tobacco hornwormManduca sexta binds to lipopolysaccharide and lipoteichoic acid. InsectMolecular Biology 2006;15:119e28.

[38] Vargas-Albores F, Jiménez-Vega F, Söderhäll K. A plasma protein isolatedfrom brown shrimp (Penaeus californiensis) which enhances the activation of

prophenoloxidase system by b-1,3-glucan. Developmental and ComparativeImmunology 1996;20:299e306.

[39] Junkunlo K, Prachumwat A, Tangprasittipap A, Senapin S, Bavornphinyo S,Flegel TW, et al. A novel lectin domain-containing protein (LvCTLD) associ-ated with response of the whiteleg shrimp Penaeus (Litopenaeus) vannameito yellow head virus (YHV). Developmental and Comparative Immunology2012;37:334e41.

[40] Amparyup P, Sutthangkul J, Charoensapsri W, Tassanakajon A. Patternrecognition protein binds to lipopolysaccharide and b-1,3-glucan and acti-vates shrimp prophenoloxidase system. Journal of Biological Chemistry2012;287:10060e9.

[41] Duvic B, Söderhäll K. Purification and characterization of a b-1,3-glucanbinding protein from plasma of the crayfish Pacifastacus leniusculus. Jour-nal of Biological Chemistry 1990;265:9327e32.

[42] Duvic B, Söderhäll K. Purification and partial characterization of a b-1,3-glucan-binding-protein membrane receptor from blood cells of the crayfishPacifastacus leniusculus. European Journal of Biochemistry 1992;207:223e8.

[43] Duvic B, Söderhäll K. b-1,3-Glucan-binding proteins from plasma of thefresh-water crayfishes Astacus astacus and Procambarus clarkii. Journal ofCrustacean Biology 1993;13:403e8.

[44] Thörnqvist PO, Johansson MW, Söderhäll K. Opsonic activity of cell adhesionproteins and b-1,3-glucan binding proteins from two crustaceans. Develop-mental and Comparative Immunology 1994;18:3e12.

[45] Yepiz-Plascencia G, Vargas-Albores F, Jimenez-Vega F, Ruiz-Verdugo LM,Romo-Figueroa G. Shrimp plasma HDL and b-glucan binding protein (BGBP):comparison of biochemical characteristics. Comparative Biochemistry andPhysiology B. Biochemistry and Molecular Biology 1998;121:309e14.

[46] Vargas-Albores F, Jiménez-Vega F, Yepiz-Plascencia GM. Purification andcomparison of b-1,3-glucan binding protein from white shrimp (Penaeusvannamei). Comparative Biochemistry and Physiology B. Biochemistry andMolecular Biology 1997;116:453e8.

[47] Yepiz-Plascencia G, Galván TG, Vargas-Albores F, García-Bañuelos M.Synthesis of hemolymph high-density lipoprotein b-glucan binding proteinby Penaeus vannamei shrimp hepatopancreas. Marine Biotechnology (NY)2000;2:485e92.

[48] Romo-Figueroa MG, Vargas-Requena C, Sotelo-Mundo RR, Vargas-Albores F,Higuera-Ciapara I, Söderhäll K, et al. Molecular cloning of a b-glucan pattern-recognition lipoprotein from the white shrimp Penaeus (Litopenaeus) van-namei: correlations between the deduced amino acid sequence and thenative protein structure. Developmental and Comparative Immunology2004;28:713e26.

[49] Lai X, Kong J, Wang Q, Wang W, Meng X. Cloning and characterization of a b-1,3-glucan-binding protein from shrimp Fenneropenaeus chinensis. MolecularBiology Reports 2011;38:4527e35.

[50] Roux MM, Pain A, Klimpel KR, Dhar AK. The lipopolysaccharide and b-1,3-glucan-binding protein gene is up-regulated in white spot virus-infectedshrimp (Penaeus stylirostris). Journal of Virology 2002;76:7140e9.

[51] Cheng W, Liu CH, Tsai CH, Chen JC. Molecular cloning and characterization ofa pattern recognition molecule, lipopolysaccharide- and b-1,3-glucan-binding protein (LGBP) from the white shrimp Litopenaeus vannamei. Fishand Shellfish Immunology 2005;18:297e310.

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

[53] Liu F, Li F, Dong B, Wang X, Xiang J. Molecular cloning and characterization ofa pattern recognition protein, lipopolysaccharide and b-1,3-glucan bindingprotein (LGBP) from Chinese shrimp Fenneropenaeus chinensis. MolecularBiology Reports 2009;36:471e7.

[54] Lin YC, Vaseeharan B, Chen JC. Identification and phylogenetic analysis onlipopolysaccharide- and b-1,3-glucan-binding protein (LGBP) of kurumashrimp Marsupenaeus japonicus. Developmental and Comparative Immu-nology 2008;32:1260e9.

[55] Sritunyalucksana K, Lee SY, Söderhäll K. A b-1,3-glucan binding protein fromthe black tiger shrimp, Penaeus monodon. Developmental and ComparativeImmunology 2002;26:237e45.

[56] VastaGR,AhmedH,TasumiS,OdomEW,SaitoK.Biological rolesof lectins in innateimmunity:molecular and structural basis for diversity in self/non-self recognition.Advances in Experimental Medicine and Biology 2007;598:389e406.

[57] Chen C, Durrant HJ, Newton RP, Ratcliffe NA. A study of novel lectins andtheir involvement in the activation of the prophenoloxidase system in Bla-berus discoidalis. Biochemical Journal 1995;310:23e31.

[58] Yu XQ, Gan H, Kanost MR. Immulectin, an inducible C-type lectin from aninsect, Manduca sexta, stimulates activation of plasma prophenol oxidase.Insect Biochemistry and Molecular Biology 1999;29:585e97.

[59] Yu XQ, Kanost MR. Immulectin-2, a lipopolysaccharide-specific lectin froman insect, Manduca sexta, is induced in response to Gram-negative bacteria.Journal of Biological Chemistry 2000;275:37373e81.

[60] Wang XW, Zhang HW, Li X, Zhao XF, Wang JX. Characterization of a C-typelectin (PcLec2) as an upstream detector in the prophenoloxidaseactivating system of red swamp crayfish. Fish and Shellfish Immunology2011;30:241e7.

[61] Wu C, Charoensapsri W, Nakamura S, Tassanakajon A, Söderhäll I,Söderhäll K. An MBL-like protein may interfere with the activation of theproPO-system, an important innate immune reaction in invertebrates.Immunobiology 2012. http://dx.doi.org/10.1016/j.imbio.2012.02.011.

P. Amparyup et al. / Fish & Shellfish Immunology 34 (2013) 990e10011000

[62] Wang XW, Wang JX. Diversity and multiple functions of lectins in shrimpimmunity. Developmental and Comparative Immunology 2012. http://dx.doi.org/10.1016/j.dci.2012.04.009.

[63] Jiang H, Kanost MR. The clip-domain family of serine proteinases inarthropods. Insect Biochemistry and Molecular Biology 2000;30:95e105.

[64] Ross J, Jiang H, Kanost MR, Wang Y. Serine proteases and their homologs inthe Drosophila melanogaster genome: an initial analysis of sequenceconservation and phylogenetic relationships. Gene 2003;304:117e31.

[65] Satoh D, Horii A, Ochiai M, Ashida M. Prophenoloxidase-activating enzyme ofthe silkworm, Bombyx mori: purification, characterization and cDNA cloning.Journal of Biological Chemistry 1999;274:7441e53.

[66] Jiang H, Wang Y, Kanost MR. Pro-phenol oxidase activating proteinase froman insect, Manduca sexta: A bacteria-inducible protein similar to Drosophilaeaster. Proceedings of the National Academy of Sciences of the United Statesof America 1998;95:12220e5.

[67] Jiang H, Wang Y, Yu XQ, Kanost MR. Prophenoloxidase-activating proteinase-2from hemolymph of Manduca sexta: A bacteria-inducible serine proteinase con-taining two clip domains. Journal of Biological Chemistry 2003;278:3552e61.

[68] Jiang H, Wang Y, Yu XQ, Zhu Y, Kanost MR. Prophenoloxidase-activatingproteinase-3 (PAP-3) from Manduca sexta hemolymph: a clip-domain serineproteinase regulated by serpin-1J and serine proteinase homologs. InsectBiochemistry and Molecular Biology 2003;33:1049e60.

[69] Gupta S, Wang Y, Jiang H. Manduca sexta prophenoloxidase (proPO) activa-tion requires proPO-activating proteinase (PAP) and serine proteinasehomologs (SPHs) simultaneously. Insect Biochemistry and Molecular Biology2005;35:241e8.

[70] Lee SY, Kwon TH, Hyun JH, Choi JS, Kawabata SI, Iwanaga S, et al. In vitroactivation of pro-phenol-oxidase by two kinds of pro-phenol-oxidase-activating factors isolated from hemolymph of coleopteran, Holotrichia dio-mphalia larvae. European Journal of Biochemistry 1998;254:50e7.

[71] Lee SY, Cho MY, Hyun JH, Lee KM, Homma KI, Natori S, et al. Molecularcloning of cDNA for pro-phenol-oxidase-activating factor I, a serine proteaseis induced by lipopolysaccharide or 1,3-b-glucan in coleopteran insect,Holotrichia diomphalia larvae. European Journal of Biochemistry 1998;257:615e21.

[72] Kwon TH, Kim MS, Choi HW, Joo CH, Cho MY, Lee BL. A masquerade-likeserine proteinase homologue is necessary for phenoloxidase activity in thecoleopteran insect, Holotrichia diomphalia larvae. European Journal ofBiochemistry 2000;267:6188e96.

[73] Lee KY, Zhang R, Kim MS, Park JW, Park HY, Kawabata S, Lee BL. A zymogenform of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca2þ in coleopteran and Tenebrio molitor larvae.European Journal of Biochemistry 2002;269:4375e83.

[74] Wang R, Lee SY, Cerenius L, Söderhäll K. Properties of the prophenoloxidaseactivating enzyme of the freshwater crayfish, Pacifastacus leniusculus. Euro-pean Journal of Biochemistry 2001;268:895e902.

[75] 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. Developmentaland Comparative Immunology 2009;33:811e20.

[76] Charoensapsri W, Amparyup P, Hirono I, Aoki T, Tassanakajon A. PmPPAE2,a new class of crustacean prophenoloxidase (proPO)eactivating enzyme andits role in PO activation. Developmental and Comparative Immunology 2011;35:115e24.

[77] 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 Immunology2011;12:70.

[78] Amparyup P, Wiriyaukaradecha K, Charoensapsri W, Tassanakajon A. A clipdomain serine proteinase plays a role in antibacterial defense but is notrequired for prophenoloxidase activation in shrimp. Developmental andComparative Immunology 2010;34:168e76.

[79] Ren Q, Xu ZL, Wang XW, Zhao XF, Wang JX. Clip domain serine proteaseand its homolog respond to Vibrio challenge in Chinese whiteshrimp, Fenneropenaeus chinensis. Fish and Shellfish Immunology 2009;26:787e98.

[80] 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. Fishand Shellfish Immunology 2011;30:980e5.

[81] Liu H, Jiravanichpaisal P, Cerenius L, Lee BL, Söderhäll I, Söderhäll K. Phe-noloxidase is an important component of the defense against Aeromonashydrophila infection in a crustacean, Pacifastacus leniusculus. Journal of Bio-logical Chemistry 2007;282:33593e8.

[82] Kim MS, Baek MJ, Lee MH, Park JW, Lee SY, Söderhäll K, et al. A new easter-type serine protease cleaves a masquerade-like protein during proph-enoloxidase activation in Holotrichia diomphalia larvae. Journal of BiologicalChemistry 2002;277:39999e40004.

[83] Liu H, Wu C, Matsuda Y, Kawabata S, Lee BL, Söderhäll K, et al. Peptidoglycanactivation of the proPO-system without a peptidoglycan receptor protein(PGRP)? Developmental and Comparative Immunology 2011;35:51e61.

[84] 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 and ShellfishImmunology 2007;22:535e46.

[85] Jitvaropas R, Amparyup P, Gross PS, Tassanakajon A. Functional character-ization of a masquerade-like serine proteinase homologue from the blacktiger shrimp Penaeus monodon. Comparative Biochemistry and Physiology B.Biochemistry and Molecular Biology 2009;153:236e43.

[86] Sriphaijit T, Flegel TW, Senapin S. Characterization of a shrimp serineprotease homolog, a binding protein of yellow head virus. Developmentaland Comparative Immunology 2007;31:1145e58.

[87] Rattanachai A, Hirono I, Ohira T, Takahashi Y, Aoki T. Peptidoglycan inducibleexpression of a serine proteinase homologue from kuruma shrimp (Marsu-penaeus japonicus). Fish and Shellfish Immunology 2005;18:39e48.

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

[89] Adachi K, Hirata T, Nishioka T, Sakaguchi M. Hemocyte components incrustaceans convert hemocyanin into a phenoloxidase-like enzyme.Comparative Biochemistry and Physiology B. Biochemistry and MolecularBiology 2003;134:135e41.

[90] Decker H, Jaenicke E. Recent findings on phenoloxidase activity and anti-microbial activity of hemocyanins. Developmental and Comparative Immu-nology 2004;28:673e87.

[91] Zlateva T, Di Muro P, Salvano B, Beltramini M. The o-diphenol oxidaseactivity of arthropod hemocyanin. FEBS Letters 1996;384:251e4.

[92] Lee SY, Lee BL, Söderhäll K. Processing of crayfish hemocyanin subunits intophenoloxidase. Biochemical and Biophysical Research Communications2004;322:490e6.

[93] García-Carreño FL, Cota K, Navarrete Del Toro MA. Phenoloxidase activity ofhemocyanin in whiteleg shrimp Penaeus vannamei: conversion, character-ization of catalytic properties, and role in postmortem melanosis. Journal ofAgricultural and Food Chemistry 2008;56:6454e9.

[94] Perdomo-MoralesR,Montero-AlejoV,PereraE, Pardo-RuizZ,Alonso-JiménezE.Hemocyanin-derivedphenoloxidase activity in the spiny lobster Panulirus argus(Latreille, 1804). Biochimica et Biophysica Acta 2008;1780:652e8.

[95] Aspán A, Huang TS, Cerenius L, Söderhäll K. cDNA cloning of prophenolox-idase from the freshwater crayfish Pacifastacus leniusculus and its activation.Proceedings of the National Academy of Sciences of the United States ofAmerica 1995;92:939e43.

[96] Gollas-Galván T, Hernández-López J, Vargas-Albores F. Prophenoloxidasefrom brown shrimp (Penaeus californiensis) hemocytes. ComparativeBiochemistry and Physiology B. Biochemistry and Molecular Biology 1999;122:77e82.

[97] Sritunyalucksana K, Cerenius L, Söderhäll K. Molecular cloning and charac-terization of prophenoloxidase in the black tiger shrimp, Penaeus monodon.Developmental and Comparative Immunology 1999;23:179e86.

[98] Amparyup P, Charoensapsri W, Tassanakajon A. Two prophenoloxidases areimportant for the survival of Vibrio harveyi challenged shrimp Penaeusmonodon. Developmental and Comparative Immunology 2009;33:247e56.

[99] Adachi K, Hirata T, Nagai K, Fujio A, Sakaguchi M. Hemocyanin-relatedreactions induce blackening of freeze-thawed prawn during storage. In:Sakaguchi M, editor. More efficient utilization of fish and fisheries products.Amsterdam: Elsevier; 2004. p. 317e30.

[100] Masuda T, Otomo R, Kuyama H, Momoji K, Tonomoto M, Sakai S, et al.A novel type of prophenoloxidase from the kuruma prawn Marsupenaeusjaponicus contributes to the melanization of plasma in crustaceans. Fish andShellfish Immunology 2012;32:61e8.

[101] Lai CY, Cheng W, Kuo CM. Molecular cloning and characterisation ofprophenoloxidase from haemocytes of the white shrimp, Litopenaeus van-namei. Fish and Shellfish Immunology 2005;18:417e30.

[102] Wang YC, Chang PS, Chen HY. Tissue expressions of nine genes important toimmune defence of the Pacific white shrimp Litopenaeus vannamei. Fish andShellfish Immunology 2007;23:1161e77.

[103] Ai HS, Huang YC, Li SD, Weng SP, Yu XQ, He JG. Characterization ofa prophenoloxidase from hemocytes of the shrimp Litopenaeus vannameithat is down-regulated by white spot syndrome virus. Fish and ShellfishImmunology 2008;25:28e39.

[104] Ai HS, Liao JX, Huang XD, Yin ZX, Weng SP, Zhao ZY, et al. A novel proph-enoloxidase 2 exists in shrimp hemocytes. Developmental and ComparativeImmunology 2009;33:59e68.

[105] Gao H, Li F, Dong B, Zhang Q, Xiang J. Molecular cloning and characterisationof prophenoloxidase (ProPO) cDNA from Fenneropenaeus chinensis and itstranscription injected by Vibrio anguillarum. Molecular Biology Reports2009;36:1159e66.

[106] Lee WJ, Ahmed A, della Torre A, Kobayashi A, Ashida M, Brey PT. Molecularcloning and chromosomal localization of a prophenoloxidase cDNA from themalaria vector Anopheles gambiae. Insect Mol Biol 1998;7:41e50.

[107] Müller HM, Dimopoulos G, Blass C, Kafatos FC. A hemocyte-like cell lineestablished from the malaria vector Anopheles gambiae expresses sixprophenoloxidase genes. Journal of Biological Chemistry 1999;274:11727e35.

[108] Christophides GK, Zdobnov E, Barillas-Mury C, Birney E, Blandin S, Blass C,et al. Immunity-related genes and gene families in Anopheles gambiae.Science 2002;298:159e65.

[109] Jiravanichpaisal P, Puanglarp N, Petkon S, Donnuea S, Söderhäll I,Söderhäll K. Expression of immune-related genes in larval stages of the gianttiger shrimp Penaeus monodon. Fish and Shellfish Immunology 2007;23:815e24.

P. Amparyup et al. / Fish & Shellfish Immunology 34 (2013) 990e1001 1001

[110] Liu Y, Li F, Wang B, Dong B, Zhang X, Xiang J. A serpin from Chinese shrimpFenneropenaeus chinensis is responsive to bacteria and WSSV challenge. Fishand Shellfish Immunology 2009;26:345e51.

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

[112] Angthong P, Watthanasurorot A, Klinbunga S, Ruangdej U, Söderhäll I,Jiravanichpaisal P. Cloning and characterization of a melanization inhibitionprotein (PmMIP) of the black tiger shrimp, Penaeus monodon. Fish andShellfish Immunology 2010;29:464e8.

[113] De Gregorio E, Han SJ, Lee WJ, Baek MJ, Osaki T, Kawabata S, et al. Animmune-responsive serpin regulates the melanization cascade in Drosophila.Developmental Cell 2002;3:581e92.

[114] Ligoxygakis P, Pelte N, Ji C, Leclerc V, Duvic B, Belvin M, et al. A serpin mutantlinks Toll activation to melanization in the host defence of Drosophila. EMBOJournal 2002;21:6330e7.

[115] Scherfer C, Tang H, Kambris Z, Lhocine N, Hashimoto C, Lemaitre B.Drosophila Serpin-28D regulates hemolymph phenoloxidase activity andadult pigmentation. Developmental Biology 2008;323:189e96.

[116] Tang H, Kambris Z, Lemaitre B, Hashimoto C. A serpin that regulates immunemelanization in the respiratory system of Drosophila. Developmental Cell2008;15:617e26.

[117] Ahmad ST, Sweeney ST, Lee JA, Sweeney NT, Gao FB. Genetic screen identifiesserpin5 as a regulator of the toll pathway and CHMP2B toxicity associatedwith frontotemporal dementia. Proceedings of the National Academy ofSciences of the United States of America 2009;106:12168e73.

[118] Michel K, Budd A, Pinto S, Gibson TJ, Kafatos FC. Anopheles gambiae SRPN2facilitates midgut invasion by the malaria parasite Plasmodium berghei.EMBO Reports 2005;6:891e7.

[119] Michel K, Suwanchaichinda C, Morlais I, Lambrechts L, Cohuet A, Awono-Ambene PH, et al. Increased melanizing activity in Anopheles gambiae doesnot affect development of Plasmodium falciparum. Proceedings of theNational Academy of Sciences of the United States of America 2006;103:16858e63.

[120] Zhu Y, Wang Y, Gorman MJ, Jiang H, Kanost MR. Manduca sexta serpin-3regulates prophenoloxidase activation in response to infection by

inhibiting prophenoloxidase activating proteinase. Journal of BiologicalChemistry 2003;278:46556e64.

[121] Tong Y, Jiang H, Kanost MR. Identification of plasma proteases inhibited byManduca sexta serpin-4 and serpin-5 and their association with componentsof the prophenoloxidase activation pathway. Journal of Biological Chemistry2005;280:14932e42.

[122] Zou Z, Jiang H. Manduca sexta serpin-6 regulates immune serine proteinasesPAP-3 and HP8: cDNA cloning, protein expression, inhibition kinetics, andfunction elucidation. Journal of Biological Chemistry 2005;280:14341e8.

[123] Jiang R, Kim EH, Gong JH, Kwon HM, Kim CH, Ryu KH, et al. Three pairs ofprotease-serpin complexes cooperatively regulate the insect innate immuneresponses. Journal of Biological Chemistry 2009;284:35652e8.

[124] Liang Z, Sottrup-Jensen L, Aspán A, Hall M, Söderhäll K. Pacifastin, a novel155-kDa heterodimeric proteinase inhibitor containing a unique transferringchain. Proceedings of the National Academy of Sciences of the United Statesof America 1997;94:6682e7.

[125] Zhao M, Söderhäll I, Park JW, Ma YG, Osaki T, Ha NC, et al. A novel 43-kDaprotein as a negative regulatory component of phenoloxidase-inducedmelanin synthesis. Journal of Biological Chemistry 2005;280:24744e51.

[126] Söderhäll I, Wu C, Novotny M, Lee BL, Söderhäll K. A novel protein acts asa negative regulator of prophenoloxidase activation and melanization in thefreshwater crayfish Pacifastacus leniusculus. Journal of Biological Chemistry2009;284:6301e10.

[127] Leclerc V, Pelte N, El Chamy L, Martinelli C, Ligoxygakis P, Hoffmann JA, et al.Prophenoloxidase activation is not required for survival to microbial infec-tions in Drosophila. EMBO Reports 2006;7:231e5.

[128] Schnitger AK, Kafatos FC, Osta MA. The melanization reaction is not requiredfor survival of Anopheles gambiae mosquitoes after bacterial infections.Journal of Biological Chemistry 2007;282:21884e8.

[129] Yeh MS, Lai CY, Liu CH, Kuo CM, Cheng W. A second proPO present in whiteshrimp Litopenaeus vannamei and expression of the proPOs during a Vibrioalginolyticus injection, molt stage, and oral sodium alginate ingestion. Fishand Shellfish Immunology 2009;26:49e55.

[130] Fagutao FF, Koyama T, Kaizu A, Saito-Taki T, Kondo H, Aoki T, et al. Increasedbacterial load in shrimp hemolymph in the absence of prophenoloxidase.FEBS Journal 2009;276:5298e306.