Developmental and Comparative Immunology 41 (2013) 597–607

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Developmental and Comparative Immunology

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A serine proteinase PmClipSP2 contributes to prophenoloxidase systemand plays a protective role in shrimp defense by scavenginglipopolysaccharide

0145-305X/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.dci.2013.06.013

⇑ Corresponding author. Tel.: +66 2 218 5439; fax: +66 2 218 5414.E-mail address: anchalee.k@chula.ac.th (A. Tassanakajon).

1 These two authors contributed equally to this work, and share the firstauthorship.

Piti Amparyup a,b,1, Kanyanat Promrungreang a,1, Walaiporn Charoensapsri a, Jantiwan Sutthangkul a,Anchalee Tassanakajon a,⇑a Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok10330, Thailandb National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Paholyothin Road, Klong1, Klong Luang,Pathum Thani 12120, Thailand

a r t i c l e i n f o a b s t r a c t

Article history:Received 13 May 2013Revised 18 June 2013Accepted 22 June 2013Available online 28 June 2013

Keywords:Shrimp immunityPenaeus monodonSerine proteinaseProphenoloxidasePattern recognition proteinHemocyte homeostasis

Serine proteinases (SPs) participate in various biological processes and play vital role in immunity. In thisstudy, we investigated the function of PmClipSP2 from shrimp Penaeus monodon in defense against bac-terial infection. PmClipSP2 was identified as a clip-domain SP and its mRNA increased in response toinfection with Vibrio harveyi. PmClipSP2-knockdown shrimp displayed a significantly reduced phenoloxi-dase (PO) activity and increased susceptibility to V. harveyi infection. Injection of LPS and/or b-1,3-glucaninduced a dose-dependent mortality and a significant decrease in the number of total hemocytes, withclear morphological changes in the hemocyte surface, of the PmClipSP2-knockdown shrimp. Recombi-nant PmClipSP2 was shown to bind to LPS and b-1,3-glucan and to activate PO activity. These resultsreveal that PmClipSP2 acts as a pattern-recognition protein, binding to microbial polysaccharides andlikely activating the proPO system, whilst it may play an essential role in the hemocyte homeostasisby scavenging LPS and neutralizing its toxicity.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Invertebrates utilize their innate immunity to protect them-selves from pathogenic infections in which the phenoloxidase(PO)-mediated melanization is a unique and effective defensemechanism that is essential for the sequestration of a variety ofmicrobial pathogens. The prophenoloxidase (proPO)-activatingsystem is controlled by the key enzyme PO that in turn is tightlyregulated in a highly elaborate manner (Cerenius and Söderhäll,2004). Activated PO catalyzes the enzymatic conversion of mono-phenolic and diphenolic substances to quinones, which undergofurther reactions to produce cytotoxic intermediates and melaninthat ultimately encapsulates and demolishes certain pathogens.Spontaneous activation of the melanization reaction needs to betightly controlled, otherwise excessive quinone as well as otherreactive intermediates produced during systemic hyperactivationof the proPO system would be deleterious to the host cells(Cerenius et al., 2008; Nappi and Christensen, 2005).

Recognition of invariant molecular patterns found in foreignpathogens, known as pathogen-associated molecular patterns(PAMPs), such as lipopolysaccharide (LPS) from Gram-negativebacteria, peptidoglycan (PGN) from Gram-positive bacteria andb-1,3-glucan from fungi, by the so-called pattern-recognition pro-teins (PRPs) is an essential step for the initiation of the proteinasecascade of the proPO system that leads to the eventual activationof proPO into PO. Proteolytic activation of the zymogen proPO ismediated by a complex cascade of clip-domain serine proteinases(clip-SPs) in which the terminal clip-SP that cleaves and activatesproPO is named the proPO-activating enzyme (PPAE) (Amparyupet al., 2013; Cerenius et al., 2008; Söderhäll et al., 2013).

Serine proteinase (SP) cascade systems play essential roles inunique biological processes, including in response to pathogenicinfections. The horseshoe crabs’ (Tachypleus tridentatus and Limuluspolyphemus) coagulation system is one of the best characterized SPcascades in arthropods (Theopold et al., 2004). Two clip-SPs (factorB and a proclotting enzyme) and two other proteinases (factors Cand G) are involved in the proteolytic cascade that leads to the for-mation of a physical barrier at the site of infection. In this case, theSP zymogen factors C and G also independently serve as PRPs forthe respective bacterial LPS and fungal b-1,3-glucan (Ariki et al.,2004; Takaki et al., 2002). In Drosophila, the SP cascade consisting

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of two clip-SPs, melanization protease-1 (MP1) and MP2, has beenreported to be involved in activation of the proPO cascade (Castil-lejo-López and Häcker, 2005; Tang et al., 2006). In Tenebrio molitor,three SPs (modular serine proteinase (MSP), spätzle-processing en-zyme-activating enzyme (SAE) and spätzle-processing enzyme(SPE)) have been shown to be important in the sequential initiationof the proPO system, which then finally cleaves SP homologue 1(SPH1) and proPO to generate a stable melanization complex forthe synthesis of melanin (Kan et al., 2008; Kim et al., 2008; Parket al., 2007). In Manduca sexta, two SP cascades have been reportedto be involved in activation of the proPO pathways. The first com-prises of the hemolymph proteinase 14 (HP14), HP21, proPO-acti-vating proteinase 2 (PAP2) and PAP3 (Gorman et al., 2007; Wangand Jiang, 2007), whilst the second cascade is involved in the acti-vation of two SPs (HP6 and PAP1) (An et al., 2009). In addition tothe activation of the SP cascade, zymogen HP14 also acts as aPRP in pathogen recognition. Autocatalysis of HP14 is inducedeither by forming a complex with b-1,3-glucans and b-1,3-glucanrecognition protein-2 (bGRP2) or by binding directly to bacterialPGNs without any other binding protein (Ji et al., 2004; Wangand Jiang, 2006). In crustaceans, several clip-SPs functioning inthe activation of the proPO system have been reported, includingone PPAE from the freshwater crayfish Pacifastacus leniusculus(Wang et al., 2001), two PPAEs from the black tiger shrimp Penaeusmonodon (Charoensapsri et al., 2009, 2011), and one PPAE from thewhite shrimp Litopenaeus vannamei (Jang et al., 2011). In addition,in the crustacean P. leniusculus, the mannose-binding lectin(Pl-MBL), a C-type lectin, has been shown to act as a PRP for LPSand also to function as a scavenger for LPS and so prevent LPSspreading in the hemolymph and avoid spatially (systemic) ortemporally extended activation of the proPO-system (Wu et al.,2013).

A number of studies have shown that the proPO system is ofbiological relevance towards a variety of pathogenic microbes.RNAi-mediated gene suppression (knockdown) of proPO tran-scripts in P. leniusculus suggested a crucial role of the proPO systemin the defense against the pathogenic bacterium Aeromonas hydro-phila (Liu et al., 2007). In the kuruma shrimp Marsupenaeus japoni-cus, silencing of the proPO gene resulted in an increased bacterialload in the shrimp hemolymph, even in the absence of an inducedbacterial or viral infection, a reduction in the circulating totalhemocyte counts (THC) and, eventually, a sharp increase in theirmortality (Fagutao et al., 2009). Silencing of the PPAE gene(LvPPAE1) in L. vannamei reduced the shrimp survival level afterVibrio harveyi challenge (Jang et al., 2011).

Previously, several proPO system components were identifiedand characterized, including two proPOs (PmproPO1 and Pmpro-PO2) and two PPAEs (PmPPAE1 and PmPPAE2), from P. monodon.Using RNAi-mediated gene knockdown, the important role of thesetwo proPOs and two PPAEs in the proPO system, as well as in theshrimp’s immune defense against the highly pathogenic bacteria,V. harveyi, was demonstrated (Amparyup et al., 2009; Char-oensapsri et al., 2009, 2011). The P. monodon LPS and b-1,3-glucanbinding protein (PmLGBP) was then subsequently characterized asa PRP for LPS and b-1,3-glucan in activation of the shrimp proPOsystem (Amparyup et al., 2012). Additionally, PmClipSP1, a homo-logue of clip-SP, has been identified as a component of shrimp anti-bacterial defense system, but does not implicated in activation of P.monodon proPO system (Amparyup et al., 2010). Here, we reportthe identification and functional characterization of PmClipSP2, anovel clip-SP from P. monodon. The transcript expression level inresponse to V. harveyi infection, the role in shrimp immune recog-nition as well as the potential function in activation of the shrimpproPO system and hemocyte homeostasis were then evaluated toaddress if PmClipSP2 has a significant role in the P. monodon im-mune system and homeostasis.

2. Materials and methods

2.1. Materials

LPS (Escherichia coli 0111:B4) and laminarin (b-1,3-glucan fromLaminaria digitata) were purchased from Sigma, whilst soluble Lys-type PGN (from Staphylococcus aureus) was purchased from Inviv-oGen. Juvenile shrimp (10–15 g) were obtained from the ShrimpGenetic Improvement Center, BIOTEC, Thailand. Other reagentswere from the sources given in the text, or were locally purchasedreagent grade (unless stated to be HPLC or Analar grade in the text)and used without further purification.

2.2. RNA extraction and cDNA synthesis

Total RNA was extracted using the TRI REAGENT� (MolecularResearch Center) according to the manufacturer’s protocol. cDNAwas synthesized using the Reverse Transcriptase System kit (Pro-mega) as per the manufacturer’s instructions and stored at�80 �C until needed.

2.3. Rapid amplification of cDNA end (RACE) and sequencecharacterization

The partial gene sequence of PmClipSP2, which exhibited a50% amino acid sequence similarity to the melanization protease1 of Drosophila, was obtained from the P. monodon EST libraryfollowing TBLASTX searching of this library using the knownshrimp PPAE sequences (Charoensapsri et al., 2009, 2011) asqueries. To obtain the full-length cDNA sequence, the 50 and 30

end RACE reactions were performed with hemocyte RNA usingthe SMART™ RACE cDNA Amplification Kit (Clontech). ThePmClipSP2 cDNA was amplified using the specific primers ofPmClipSP2R for the 50 RACE and PmClipSP2F for the 30 RACE(Table S1), according to the manufacturer’s instructions, underthe following conditions: 94 �C for 1 min; followed by 25 cyclesof 94 �C for 30 s, 68 �C for 30 s and 72 �C for 3 min. The expectedPCR fragments from the 50 RACE and 30 RACE reactions werecloned using the TA cloning system and sequenced. To amplifythe single PCR fragment, RT-PCR was performed with the primerpair 5PmClipSP2-F and 3PmClipSP2-R (Table S1) with Pfu DNApolymerase (Promega). The obtained PCR product was thencloned and sequenced.

The nucleotide sequence was analyzed using the Genetyx soft-ware. The deduced protein sequence was compared to the proteindatabase using the BLASTP program at the National Center for Bio-technology Information (http://www.ncbi.nlm.nih.gov/BLAST/).The putative protein domains were predicted by the simple modu-lar architecture research tool (SMART) software (http://smar-t.embl-heidelberg.de/). Multiple alignments were performedusing the ClustalW2 program (http://www.ebi.ac.uk/Tools/clu-stalw2/).

2.4. Tissue expression analysis of PmClipSP2

In order to investigate the tissue expression of PmClipSP2, semi-quantitative RT-PCR was conducted using the SP2-F/-R primer pair(Table S1) on the cDNA from the hemocytes, hepatopancreas, lym-phoid organ, intestine, heart and gill, each obtained from thepooled RNA from the respective tissue type from three healthyshrimp. The expression levels were normalized relative to that ofthe elongation factor 1-a (EF1-a) gene.

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2.5. Real-time RT-PCR analysis of PmClipSP2 transcript levels inresponse to challenge with V. harveyi

Quantitative real-time RT-PCR analysis using an iCycler-iQ™system (Bio-Rad Laboratories) was applied to determine thetranscript expression profiles of PmClipSP2 in P. monodon hemo-cytes in response to infection with the pathogenic Gram-nega-tive bacteria V. harveyi 639. Total RNA from hemocytes wereextracted at 0, 3, 6, 12, 24 and 48 h post-infection (hpi) fromthree shrimp per treatment and time point and pooled. ThecDNA was then synthesized from each total RNA preparationusing the cDNA synthesis kit (QIAGEN). Real-time PCR analysiswas performed as described by Amparyup et al. (2007) usingthe PmClipSP2F/R primer pair (Table S1), and EF1-a was ampli-fied as the internal control and reference standard to verify thequantitative real time PCR reaction. Each sample pooled fromthree shrimp at each time point was measured three times.The Ct values of the infected hemocyte samples at each timepoint were normalized with the saline-injected samples andthe mathematical model of Pfaffl was used to determine the rel-ative expression ratio (Pfaffl, 2001).

2.6. In vivo gene knockdown of PmClipSP2

To generate a PmClipSP2 template for dsRNA synthesis, the DNAfragment was amplified by PCR from a full-length PmClipSP2 con-taining plasmid using the sense and anti-sense PmClipSP2-specificprimers (Table S1) flanked by the T7 promoter site. In addition, aGFP fragment from the pEGFP-1 plasmid (Clontech) was producedby PCR with specific primers (Table S1). The respective PCR prod-uct was used as template to synthesize dsRNAs in vitro using theT7 RiboMAX™ Express Large Scale RNA Production Systems (Pro-mega) following the manufacturer’s protocol to obtain the dsRNAfor PmClipSP2 and GFP.

RNAi-mediated gene knockdown was achieved by double-injec-tions of 2.5 lg dsRNA/g of shrimp wet body weight in normal ster-ile saline (NSS; 150 mM NaCl) as previously described (Amparyupet al., 2009). Shrimp injected with the GFP dsRNA in NSS at thesame concentration or NSS only were served as control groups.After the 2nd dsRNA (or NSS) injection, the shrimp were rearedfor a further 48 h prior to RT-PCR analysis. The hemolymph fromeach individual shrimp was collected without anticoagulant andsubjected to RNA isolation using the RNA isolation kit (GE Health-care). Semi-quantitative RT-PCR analysis of PmClipSP2 transcriptlevels in three individual shrimp from each experiment was con-ducted at 48 h post-injection as described above to ascertain thereduction in the transcript levels.

2.7. PO activity assay of knockdown shrimp

The PO activity was assayed using the method of Amparyupet al. (2009). In brief, the whole hemolymph was withdrawn fromeach shrimp at 48 h after the 2nd dsRNA or NSS injection. Hemo-lymph protein (2 mg), determined by a Bradford assay kit (Bio-Rad), was added into 10 mM of Tris–HCl, pH 8.0 to final volumeof 435 ll and then mixed with 65 ll of 3 mg/ml L-3,4-dihydroxy-phenylalanine (L-DOPA). After incubation for 30 min, the reactionwas stopped by adding 500 ll of 10% (v/v) acetic acid and the POactivity was recorded as the DA490/mg total protein/min. Eachexperimental group (three shrimp/group) was performed in tripli-cate. Data were analyzed using one-way analysis of variance (AN-OVA) followed by Duncan’s test and statistical significance wasaccepted at P < 0.05.

2.8. Shrimp survival assay

To test the potential importance of PmClipSP2 in the immunedefense, the survival of PmClipSP2 knockdown shrimp was deter-mined. Three groups of shrimp were intramuscularly injected with25 ll of NSS alone or containing 2.5 lg/g shrimp of PmClipSP2dsRNA or GFP dsRNA, as described above. For the 2nd dsRNA orNSS injection, shrimp were injected as before except the 25 llinjection additionally contained 2 � 105 colony forming units(CFUs) of V. harveyi 639. The number of surviving shrimp survivalwas recorded every 6 hpi and thereafter up to 120 hpi with V.harveyi. Each experiment (10 healthy shrimp/group) was per-formed in triplicate.

For analysis of the hemolymph total viable bacterial counts, thehemolymph of PmClipSP2 knockdown shrimp was collected at6 hpi and diluted in PBS buffer (pH 7.4). Serial dilutions of thehemolymph were then plated on LB agar, followed by incubationat 30 �C overnight prior to counting individual colonies and deter-mination of the bacterial load as CFU/ml. Data from the triplicateexperiments were analyzed using one-way ANOVA followed byDuncan’s test.

2.9. LPS- and b-1,3-glucan- induced lethality in PmClipSP2 knockdownshrimp, compared to that in PmPPAE2 and PmLGBP knockdownshrimp

Preliminary results indicated that control shrimp were typicallyresistant to LPS or laminarin (b-1,3-glucan) at up to 50 lg/g ofshrimp. However, surprisingly the PmClipSP2 knockdown shrimpwere far more sensitive to LPS or to b-1,3-glucan. Thus, the lethal-ity of LPS or b-1,3-glucan in PmClipSP2 knockdown shrimp wasexamined. The intramuscular injection of 1, 5 or 10 lg/g shrimpof LPS or b-1,3-glucan was performed at 24 h after the 2nd injec-tion of dsRNA (PmClipSP2 or GFP) or NSS. The same procedurewas also performed for the knockdown of PmPPAE2 and PmLGBP,which are the respective clip-SP and a PRP that previously foundto function in the proPO system of shrimp P. monodon. Lethalitywas determined at 3 h to 72 h after LPS and laminarin injectionand expressed in terms of the mean percentage survival and statis-tical analysis was performed using the one-way ANOVA followedby Duncan’s test.

2.10. LPS effects on PmClipSP2 knockdown shrimp hemocytes in vivo

To determine the effect of LPS on hemocyte homeostasis,shrimp were injected with 10 lg/g shrimp LPS after the 2nd injec-tion of PmClipSP2 or GFP dsRNA. One hour after LPS injection, 50 llof hemolymph was withdrawn from three to five shrimp and fixedwith 20% (w/v) formalin in PBS buffer (pH 7.4). The THC was thenevaluated with a hemocytometer under a light microscope, and theaverage THC of each group was calculated. The experiment wasperformed in triplicate and statistical analysis was performedusing the one-way ANOVA followed by Duncan’s test.

2.11. SEM analysis of the hemocyte morphology

In order to monitor the effect of LPS on the hemocytes of thePmClipSP2 knockdown shrimp, shrimp were injected twice withdsRNA (PmClipSP2 or GFP), the second injection being with orwithout LPS (10 lg/g shrimp) as detailed above. Hemolymph wassubsequently collected at 1 h after the second injection and fixedwith 2.5% (w/v) glutaraldehyde in 0.1 M phosphate buffer (pH7.4) on a glass coverslip for 2 h at room temperature. Cells werethen washed three times with 0.1 M phosphate buffer pH 7.4 anddehydrated stepwise by exposure to the ethanol series and criticalpoint dried in liquid carbon dioxide. Samples were then mounted

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on specimen stubs and sputter coated with gold before beingobserved under a JEOL scanning electron microscope (modelJSM-5410LV) at an accelerating voltage of 15 kV.

2.12. Construction and expression of recombinant (r) PmClipSP2protein

The mature PmClipSP2 encoding DNA fragment (Accession No.FJ620687) flanked by PciI and XhoI restriction enzyme sites was ob-tained via PCR using Pfu DNA polymerase (Promega) and the SP2m-F/-R primers (Table S1). This was then cloned into the pET28bexpression vector, as a N-terminal (His)6 tagged encoding chimera,and transformed into E. coli JM109 cells and recombinant transfor-mants selected by growth on LB-ampicillin (100 lg/ml) plates. Thepurified recombinant plasmid was confirmed for the correct insertand in-frame position by DNA sequencing and then transformedinto E. coli Rosetta (DE3)-pLysS cells (Novagen) for expression. Re-combinant PmClipSP2 expression was induced with 1 mM IPTG for5 h at 37 �C in a shaking incubator (250 rpm), and then purifiedand refolded as described previously (Amparyup et al., 2008).The protein preparation was then concentrated though a Microcon10 (Millipore). The protein concentration was determined by theBradford assay and stored as aliquots at �80 �C. To test forrPmClipSP2 expression and its apparent purification, the proteinpreparation was resolved per lane on duplicate SDS–PAGE gelsand one set stained with Coomassie Brilliant Blue to visualize theprotein bands, and the other set electro-transferred to PVDF mem-brane (GE Healthcare) for western blot detection of the rPmClipSP2using the mouse anti-His tag monoclonal antibody (GenScript) orrabbit polyclonal anti-PmClipSP2 antibody, as described by Amp-aryup et al. (2012).

2.13. ELISA-based binding assay of rPmClipSP2

Binding of rPmClipSP2 to PAMPs was performed as previouslydescribed by Amparyup et al. (2012). Briefly, LPS, b-1,3-glucan(laminarin) or soluble Lysine (Lys)-type PGN were coated over-night in 96-well microtiter plates (Costar) at 37 �C. After incubat-ing for 1 h at 60 �C, wells were blocked with Tris buffer solution(TBS) containing 0.1% (w/v) BSA for 2 h before being incubatedwith 100 ll of rPmClipSP2 (0–10 lM in TBS) at room temperaturefor 3 h. The wells were then washed with TBS containing 0.1% (v/v) Tween 20 (TBST) and the bound protein was detected immuno-chemically using a mouse anti-His tag monoclonal antibody fol-lowed by incubation with alkaline phosphatase-conjugatedrabbit anti-mouse IgG as a secondary antibody. Binding was de-tected by the alkaline phosphatase reaction, as described previ-ously (Amparyup et al., 2012). All assays were performed intriplicate, and wells with the addition of TBS without anyrPmClipSP2 protein were used as the negative control. Statisticaltests were performed using GraphPad Prism� 5 software (Graph-Pad Software, Inc.), where the data were fitted with a one-sitebinding model and the Kd value was determined from the non-linear curve fitting as A = Amax [L]/(Kd + [L]), where A is the absor-bance, which is proportion to the bound concentration, Amax isthe maximum binding and [L] is the concentration of therPmClipSP2 protein.

2.14. Activation of PO activity by rPmClipSP2

To evaluate whether rPmClipSP2 can trigger the activation ofshrimp proPO system in vitro, PO activity assays were performedas described previously (Amparyup et al., 2012). In brief, 250 lgof hemolymph protein from a healthy shrimp dissolved in10 mM Tris–HCl (pH 8.0) was incubated in the presence or absenceof 1.0 lg/ml LPS or laminarin and 20 lM rPmClipSP2.

Subsequently, L-DOPA (3 mg/ml) was added to each reaction andincubated at room temperature for 30 min. The absorbance at490 nm was then measured using a spectrophotometer.

For the in vivo assay, shrimp were injected with rPmClipSP2protein (10 lg/g shrimp) or with BSA (10 lg/g shrimp) as a control.Shrimp hemolymph was then collected from five individual in-jected shrimp from each group without anticoagulant at 24 hpost-injection. Activation of PO activity was measured spectropho-tometrically using L-DOPA as described above.

3. Results

3.1. PmClipSP2 likely belongs to the family of arthropod clip-SPs

To identify clip-SPs in the P. monodon shrimp proPO system,we used the known full-length PPAE shrimp sequences(Charoensapsri et al., 2009, 2011) to query the P. monodon ESTdatabase (http://pmonodon.biotec.or.th) using the TBLASTX algo-rithm. From this search PmClipSP2, which displayed the highestpredicted amino acid sequence similarity to the melanization pro-tease of Drosophila melanogaster (50% similarity), was identified. Afull-length cDNA of PmClipSP2 (GenBank accession number:FJ620687) was then obtained by RACE analysis and found to bea 1330-nucleotide sequence including a 17-base poly(A) tail.The predicted open reading frame encoded for a deduced 27-ami-no acid signal peptide and 342-amino acid mature protein. Thededuced protein sequence was then used to perform a primarystructure-based search using SMART analysis, revealing that themature peptide contains a clip domain at the N-terminus and atrypsin-like SP domain at the C-terminus (Fig. 1A). Interestingly,homology searching of the NCBI GenBank database showed thisprotein sequence exhibited the highest sequence similarity toan array of crustacean (50–90% similarities) and insect (50–55%similarities) PPAEs, examples of which are shown in the multiplesequence alignment in Fig. S1.

To further verify the structural features of PmClipSP2, we car-ried out a structural alignment of PmClipSP2 to other knownclip-SP sequences from crustaceans and insects. As anticipated,the essential six conserved cysteine residues in the clip domainthat form the three disulfide bonds and the three conserved cata-lytic triad residues of a typical trypsin-like SP domain were con-served in all these sequences (Fig. S1).

3.2. PmClipSP2 is expressed in shrimp hemocytes and is an immune-responsive gene

The pattern of PmClipSP2 transcript expression was examinedin order to confirm that it is a component of shrimp hemocytes.Using specific primers for PmClipSP2, the cDNA was amplifiedby RT-PCR from six different tissue types in P. monodon shrimp,revealing that PmClipSP2 transcript levels were highly abundantin hemocytes, detected at low levels in the heart, but not detectedin the hepatopancreas, gills, lymphoid organ or intestine (Fig. 1B).To determine the effect of infection with the Gram-negative bac-teria V. harveyi 639 on the PmClipSP2 transcript levels, PmClipSP2mRNA levels in the hemocytes of control and V. harveyi chal-lenged shrimp were measured by real-time quantitative RT-PCR.The highest expression level of PmClipSP2 mRNA was observedat 3 hpi at a 2.6-fold higher relative expression level than at0 hpi (Fig. 1C). By 6–12 hpi, the transcript levels had decreasedback to normal expression levels. However, by 24–48 hpi,PmClipSP2 transcript levels were dramatically decreased to belowthe control levels suggesting a transient strong down-regulationafter infection (Fig. 1C).

Fig. 1. PmClipSP2 is an immune-responsive gene in shrimp hemocytes. (A) Schematic structure illustrating the putative domains of PmClipSP2. The annotated N-terminalclip-domain and C-terminal trypsin-like SP domain are indicated. (B) Tissue expression analysis of PmClipSP2 transcripts by semi-quantitative RT-PCR. Total RNA wasextracted from the indicated six tissues of P. monodon (hemocytes, hepatopancreas, gills, lymphoid organ, intestine and heart) and RT-PCR amplification was then performedusing gene specific primers for PmClipSP2 and EF1-a (internal control). Results shown are representative of three independent trials. (C) Quantification of the time course ofPmClipSP2 mRNA expression levels in shrimp hemocytes after systemic challenge with V. harveyi. Each sample was pooled from three shrimp at each time point andmeasured tree time. The values shown are the expression level of V. harveyi-infected shrimp at each time point that relative to their internal control, EF1-a gene, andnormalized with the saline-injected sample. Data are shown as the mean ± 1 SEM, derived from triplicate samples per assay and three independent assays.

Fig. 2. PmClipSP2 is required for activation of the proPO system in shrimp. (A) Theknockdown efficiency of PmClipSP2 dsRNA in shrimp hemocytes at 48 h after the2nd dsRNA injection, as evaluated for the PmClipSP2 transcript levels by semi-quantitative RT-PCR. Shrimp injected with GFP dsRNA in NSS or NSS alone served ascontrol groups and EF1-a was used as the loading control. Each lane is represen-tative of the results obtained from each individual shrimp (n = 3). (B) PmClipSP2knockdown diminishes the PO activity in shrimp hemolymph. Hemolymph wascollected at 48 h after the 2nd dsRNA (or NSS) injection and then the PO enzymaticactivity was measured at 30 min after incubation with L-DOPA and defined asDA490/mg total protein/min. Data are shown as the mean ± 1 SEM, and are derived

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3.3. PmClipSP2 knockdown reduces the hemolymph PO activity

To test the hypothesis that PmClipSP2 is involved in the shrimpproPO system, the PmClipSP2 mRNA expression level was reducedsystemically by dsRNA-mediated gene knockdown in juvenile P.monodon shrimp. The transcript expression levels of PmClipSP2were significantly decreased in the PmClipSP2 dsRNA knockdownshrimp, when compared to that in the control GFP dsRNA or NSSinjected shrimp (Fig. 2A). The specificity and efficiency ofPmClipSP2 gene silencing, compared to the other known P. mon-odon clip-SPs (PmPPAE1, PmPPAE2 and PmClipSP1) was evaluatedas described previously (Charoensapsri et al., 2009), and found tobe effective and sequence specific for PmClipSP2 (Fig. S2). As a re-sult of the reduction in the PmClipSP2 transcript expression (�60%reduction), a significant decrease in the PO activity in the hemo-lymph was observed, being 45% of the control after 48 h(Fig. 2B). Taken together, these data suggest that PmClipSP2 func-tions in the shrimp proPO system.

Whether PmClipSP2 is required for the regulation of the expres-sion of any other proPO-related gene transcripts was evaluated byRT-PCR in the PmClipSP2 dsRNA knockdown shrimp. The injectionof PmClipSP2 dsRNA did not interfere with the transcript expres-sion levels of the three other proPO-associated genes assayed(PmproPO1, PmproPO2 and PmLGBP), whilst the correspondingdsRNA mediated gene knockdowns of these three genes did notsignificantly affect the transcript expression level of PmClipSP2(Fig. S2). The PmClipSP2, PmLGBP and PmproPO1/2 gene knock-down shrimp all showed a significantly reduced transcript levelfor the respective knockdown gene, but not those of the othergenes, except for the reduction of PmLGBP transcript levels in thePmproPO1/2 knockdown shrimp. Thus, then no significant interac-tion between PmClipSP2 mRNA expression and those of PmLGBPand PmproPO1/2 was observed.

from three independent experiments. Statistical significance between means isindicated with an asterisk (⁄⁄P < 0.01).

3.4. PmClipSP2 is a crucial molecule in shrimp immunity againstV. harveyi infection

The potential role of PmClipSP2 in the defense of P. monodonshrimp against pathogenic bacteria was evaluated using systemicinfection with V. harveyi 639 as a model bacterial pathogen. Inthe PmClipSP2 knockdown shrimp, infection with V. harveyi ledto a rapid mortality (0% survival within 24 h), which was

significantly higher than that for the control shrimp injected witheither GFP dsRNA in NSS or NSS only (Fig. 3A). In addition, theviable bacterial number in the hemolymph of the shrimp wassignificantly increased in the PmClipSP2 knockdown shrimp at 6hpi compared to the control shrimp (Fig. 3B). These resultssuggested that PmClipSP2 plays an essential role in the defenseagainst V. harveyi infection.

Fig. 3. PmClipSP2 knockdown increases the susceptibility of shrimp to V. harveyi infection in vivo. (A) Survival analysis of PmClipSP2 knockdown shrimp after V. harveyi(2 � 105 CFUs) infection. PmClipSP2 knockdown shrimp and the control groups (GFP dsRNA or NSS only) were infected with V. harveyi and the shrimp mortality was recordedevery 6 hpi. Data are shown as the mean ± 1 SEM survival rate (%), derived from three independent experiments. (B) Viable bacterial numbers in the hemolymph of PmClipSP2knockdown and control shrimp after V. harveyi infection. Hemolymph was collected from the shrimp at 6 hpi and the number of viable bacteria in the hemolymph, as colonyforming units (CFUs) per ml of shrimp hemolymph, is reported as the mean ± 1 SEM, derived from three independent experiments. Asterisk indicates the means aresignificantly different (⁄⁄⁄P < 0.001).

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3.5. Microbial cell wall components (LPS and b-1,3-glucan) are highlytoxic to PmClipSP2 knockdown shrimp

In general, microbial cell wall components, such as LPS andb-1,3-glucan, are potent stimulants of the shrimp innate immunity,but a high dose of LPS can kill shrimp in a dose dependent manner(Lorenzon et al., 1999). Previous studies have shown that the coin-jection of LPS/b-1,3-glucan (laminarin), each at 10 lg/g shrimp, didnot significantly affect the survival of any of the PmproPO1, Pmpro-PO2, PmPPAE1, PmPPAE2, PmLGBP or PmClipSP1 knockdownshrimp (one gene knockdowns, mediated by dsRNA injection)(Amparyup et al., 2009, 2010, 2012; Charoensapsri et al., 2009,2011), whilst here preliminary results revealed that shrimp wildtype (control) P. monodon were typically resistant to LPS and lam-inarin at up to 50 lg/g shrimp (data not shown). In contrast, theco-injection of LPS and b-1,3-glucan in the PmClipSP2 knockdownshrimp was found to increase their mortality rapidly within 6 to24 h after injection (Fig. 4A). Accordingly, PmClipSP2 might playa role in reducing the toxicity of LPS or b-1,3-glucan. To test thisassumption, a preliminary evaluation of the lethal effect of injec-tion with various doses of LPS and/or laminarin (b-1,3-glucan) onPmClipSP2 knockdown shrimp was performed. The mortality ofPmClipSP2 knockdown shrimp was dose-dependently increasedwith increasing levels of injected LPS (Fig. 4B) or b-1,3-glucan(Fig. 4C), with the PmClipSP2 knockdown shrimp having a greatersensitivity to LPS than to b-1,3-glucan. Although LPS was thusmore toxic than b-1,3-glucan on a weight for weight basis, thecombined treatment with both together at 10 lg/g shrimp weightresulted in a higher mortality than either LPS or b-1,3-glucan aloneat 6 and 24 h after injection onwards (but not at 12 h after injec-tion). However, it remains to be evaluated if this is synergistic ornot. Additionally, the death of PmClipSP2 knockdown shrimp afterLPS injection was not prevented by treatment with antibiotics (tet-racycline and kanamycin) (data not shown).

3.6. Involvement of PmClipSP2 in hemocyte homeostasis post LPSstimulation

In crustacean species, hemocytes are very important to immunedefense and homeostasis. Systemic exposure to LPS or b-1,3-glucanis known to induce an immediate but transient reduction in thecirculating THC in crustaceans, which then started to increase 3 hlater and returned to normal levels at 24 h after injection

(Lorenzon et al., 1999; Söderhäll et al., 2003). Given that thePmClipSP2 knockdown shrimp were found to be more sensitiveto LPS than to laminarin (b-1,3-glucan), the effect of LPS on the cir-culating THC in P. monodon was then evaluated. The injection ofLPS (10 lg/g shrimp) into the control P. monodon shrimp had nosignificant effect on the THC level, whilst although the PmClipSP2knockdown alone reduced the circulating THC numerically to70% of the control level, which was not statistically significant.However, injection of LPS into the PmClipSP2 knockdown shrimpcaused a significant (4.2-fold) reduction in the circulating THCdown to 22% of the control level (Fig. 5A and B).

Scanning electron microscope (SEM)-based analysis of thehemocyte morphology revealed a clear change in the hemocyte cellmembrane of the PmClipSP2 knockdown shrimp and severe dam-ages to the hemocytes including cell disruption, uncontrolledswelling and eventually bursting were clearly observed in thePmClipSP2 knockdown shrimp after injection with LPS (Fig. 5C).In contrast, no morphological change in the hemocytes was noticedin the control (GFP dsRNA injected) shrimp even after injectionwith LPS. Thus, PmClipSP2 may neutralize the LPS toxicity and par-ticipate in the maintenance of hemocyte homeostasis.

3.7. PmClipSP2 interacts with both LPS and laminarin (b-1,3-glucan)

To further elucidate the binding properties of PmClipSP2, re-combinant (r)PmClipSP2 was produced as a (His)6 tagged chimeraand then used to perform an ELISA based binding assay with threedifferent PAMPs (LPS, b-1,3-glucan (laminarin) and Lys-type-PGN).Surprisingly, rPmClipSP2 was found to directly bind to bothimmobilized LPS and b-1,3-glucan (Fig. 6A and B), but not withthe Lys-type PGN (data not shown). Analysis of the binding inter-actions reveals that rPmClipSP2 bound b-1,3-glucan with a 1.9-foldhigher affinity (dissociation constant (Kd) of 8.05 � 10�7 M) thanLPS (Kd of 1.56 � 10�6 M) and this high affinity binding for bothLPS and b-1,3-glucan was dose-dependent and saturable. Accord-ingly, PmClipSP2 may function as a PRP for initiating the shrimpimmune response to Gram-negative bacteria (LPS) and fungi(laminarin as a representative of b-1,3-glucan).

3.8. The proPO system activation by PmClipSP2 in the presence of LPS

At least two P. monodon proteins, PmLGBP and PmMasSPH1,have been shown previously to associate with LPS and to be

Fig. 4. PmClipSP2 knockdown shrimp are dose-dependently susceptible to LPS and b-glucan in vivo. (A) Survival rates of the PmClipSP2, PmPPAE2, PmLGBP or GFP (control)knockdown shrimp after subsequent challenge with LPS and b-glucan (right) or NSS only (left). PmClipSP2 knockdown shrimp (and the PmPPAE2, PmLGBP and GFP dsRNAs inNSS or NSS only as the control injected shrimp) were co-injected with LPS and b-1,3-glucan (each at 10 lg/g shrimp) 24 h after the 2nd dsRNA injection, and the shrimpmortality was recorded at 3 h after the second injection and daily thereafter. The data are shown as the mean ± 1 SEM percentage survival, derived from three independentexperiments. (B and C) Dose-dependent effect of (B) LPS and (C) b-1,3-glucan on the survival of PmClipSP2 knockdown shrimp. PmClipSP2 knockdown shrimp, and the fourcontrol groups (PmPPAE2, PmLGBP and GFP dsRNAs or NSS) were injected with different concentrations of either LPS or b-1,3-glucan (1, 5 or 10 lg/g shrimp) at 24 h after the2nd dsRNA or NSS injection. The shrimp mortality was then recorded at 3 h to 72 h (daily from 24 h onwards) after the LPS or b-1,3-glucan injection and is shown as themean ± 1 SEM percentage survival, derived from three independent experiments.

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involved in the activation of shrimp immunity (Amparyup et al.,2012; Jitvaropas et al., 2009). More recently, it was reported thatPmLGBP acts a PRP with both LPS and b-1,3-glucan binding activityand then activates the shrimp proPO system (Amparyup et al.,2012). In the present study, whether or not PmClipSP2 triggersthe proPO activation cascade in P. monodon was evaluated. Incuba-tion of the purified rPmClipSP2 and total hemolymph proteins of P.monodon shrimp with or without LPS and b-1,3-glucan, followedby measuring the PO activity using L-DOPA as the substrate, re-vealed that the PO activity was slightly increased numerically bythe addition of b-1,3-glucan, LPS or rPmClipSP2, but these werenot statistically significant. However, the coaddition of rPmClipSP2with either b-1,3-glucan or LPS to the hemolymph proteins signif-icantly increased the PO activity, respectively, compared with thebasal level of PO activity in the hemolymph protein (Fig. 7A andB). Moreover, direct injection of the rPmClipSP2 into shrimp alsoshowed a corresponding significant increase in the PO activity(Fig. 7C). Taken together, these in vitro and in vivo results are con-sistent with the notion that rPmClipSP2 interacts with both LPSand b-1,3-glucan and induces the proPO activation system inP. monodon shrimp.

4. Discussion

ProPO and associated proteins (PRPs, proteinases and inhibi-tors) in the melanization cascade have been characterized andstudied in a number of invertebrate species (Amparyup et al.,2013; Cerenius et al., 2008; Cerenius and Söderhäll, 2004; Kanostand Gorman, 2008), but the likely function of several putative pro-teins in the proPO system remains unknown. In crustaceans, theproPO system is reported to be important in the immune defenseagainst pathogenic bacteria and fungi (Amparyup et al., 2009,2013; Charoensapsri et al., 2009, 2011; Fagutao et al., 2009; Janget al., 2011; Liu et al., 2007). Furthermore, the recognition of path-ogen cell wall components by PRPs is a critical step in activation ofthe melanization cascade. Recent evidence in insects suggests thatcertain SPs act as PRPs and are required for the activation of themelanization cascade to recognize the microbes (Ji et al., 2004;Wang and Jiang, 2006).

SPs play a crucial role in host–pathogen interactions. The SPdomain of clip-SPs belongs to the trypsin-like SP family, whichhave been identified in both vertebrates and invertebrates. Thebiological functions of this proteinase family have been extensively

Fig. 5. PmClipSP2 participates in the maintenance of shrimp hemocyte homeostasis in the presence of LPS. (A and B) The circulating THC in the PmClipSP2 knockdown shrimpcompared to the dsRNA GFP injected control shrimp in the absence or presence of LPS (10 lg/g shrimp). Data are shown as the mean ± 1 SEM, derived from triplicateexperiments. Asterisk indicates a statistical significant difference between means (⁄P < 0.05, ⁄⁄P < 0.01). (C) SEM images of hemocytes from the PmClipSP2 knockdown shrimpcompared to the dsRNA GFP injected control shrimp in the absence or presence of LPS (10 lg/g shrimp). Images shown are representative of at least three such fields of viewper sample and three independent samples.

Fig. 6. PmClipSP2 binds to LPS and b-1,3-glucan (laminarin). ELISA assay demonstrating the dose-dependent binding of rPmClipSP2 (0–10 lM) to immobilized (A) LPS or (B)b-1,3-glucan. Solid lines illustrate the fitted curves. Data are shown as the mean ± 1 SEM, derived from triplicate experiments.

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characterized and they have been found to be involved in immu-nity and homeostasis (Jang et al., 2008; Jiang and Kanost, 2000).In this study, PmClipSP2, a clip-SP from P. monodon, was identifiedand its significant function is reported. Sequence analysis showedthat PmClipSP2 contains an N-terminus clip domain and a C-termi-nus SP domain and so is a member of the clip-SP family (Jang et al.,2008; Jiang and Kanost, 2000). PmClipSP2 was found to mainly beexpressed in hemocytes, which is in agreement with the expres-sion pattern of the other known proPO-associated transcripts inP. monodon (PmproPO1, PmproPO2, PmPPAE1, PmPPAE2 andPmLGBP) that are primarily detected in the hemocytes (Amparyupet al., 2009, 2012; Charoensapsri et al., 2009, 2011). Moreover, theexpression level of PmClipSP2 changes in response to infectionwith V. harveyi, supporting that PmClipSP2 is likely to be involvedin shrimp immune responses.

Previous studies have demonstrated that two clip-SPs,PmPPAE1 and PmPPAE2, but not PmClipSP1, are involved in theactivation of the shrimp proPO system (Amparyup et al., 2010;Charoensapsri et al., 2009, 2011). However, the role of PmClipSP2in the proPO cascade is unknown. To address this, PO activity as-

says were performed on the PmClipSP2 knockdown shrimp, and re-vealed that PmClipSP2 knockdown leads to a reduction in thehemolymph PO activity. Thus, in contrast to PmClipSP1, PmClipSP2is likely to be involved in the melanization cascade in P. monodon.The protective role of several genes (PmproPO1 and 2, PmPPAE1and 2) in the shrimp proPO system was recently verified usinggene silencing, where knockdown shrimp showed an enhancedmortality to pathogenic bacterial infection, and this was associatedwith increased bacterial loads in the hemolymph (Amparyup et al.,2009; Charoensapsri et al., 2009, 2011). In accordance, in this studyan enhanced mortality of PmClipSP2 knockdown shrimp was ob-served after infection with V. harveyi, supporting that melanizationis an important immune defense in fighting microbial infections.

In invertebrates, several proteins act as PRPs (receptors) for LPSor b-1,3-glucan (Cerenius et al., 2010; Wang and Wang, 2013). Be-cause shrimp with reduced PmClipSP2 transcript levels showed anincreased sensitivity to LPS and b-1,3-glucan, the notion that thisprotein might interact directly with these microbial cell wall com-ponents was evaluated using ELISA binding assays. The recombi-nant PmClipSP2 protein was not found to bind to PGN but bound

Fig. 7. PmClipSP2 is required for the LPS and b-1,3-glucan induced proPO system activation in shrimp hemolymph. (A and B) In vitro PO activity assay, showing the ability ofrPmClipSP2 to activate the proPO system in vitro. Hemolymph (250 lg in 10 mM Tris–HCl pH 8.0), withdrawn from a normal shrimp, was incubated with 20 lM rPmClipSP2in the absence or presence of 1.0 lg/ml of pre-incubated (A) LPS or (B) b-1,3-glucan. Controls were performed as above except without the rPmClipSP2. The PO activity wasspectrometrically measured after 30 min using L-DOPA as the substrate, as outlined in the methods. Data are shown as the mean ± 1 SEM, derived from triplicate experiments.Means with an asterisk are significantly different (⁄⁄P < 0.01). (C) Activation of the proPO system in vivo by rPmClipSP2. Shrimp were injected with 10 lg/g shrimp of eitherrPmClipSP2 or BSA in NSS, or NSS alone, and then the hemolymph was collected 24 h later and assayed for PO activity as above. Data are shown as the mean ± 1 SEM, derivedfrom triplicate experiments. Means with an asterisk are significantly different (⁄⁄P < 0.01).

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with high affinity to both LPS and b-1,3-glucan in the ELISA basedbinding assay, and to activate the proPO system upon binding toeither of them, suggesting that PmClipSP2 also acts as a PRP inthe proPO system of P. monodon.

The involvement of SPs in the recognition processes of thehorseshoe crab (L. polyphemus and T. tridentatus) coagulation cas-cade system, composed of factor C, an endotoxin-sensitive SPzymogen, and factor G, a glucan-sensitive SP zymogen, as well asin the recognition processes of the tobacco hornworm (M. sexta)proPO cascade system that includes the SP HP14 that interactswith the Gram-positive bacterial PGN or fungal b-1,3-glucan, havealso been reported (Ariki et al., 2004; Ji et al., 2004; Takaki et al.,2002; Theopold et al., 2004; Wang and Jiang, 2006). However, un-like other known SPs, which are composed of a single SP domain,factor C, factor G and HP14 contain additional distinct domains in-volved in the immune recognition of pathogens. In contrast,PmClipSP2 exhibited only the single predicted N-terminal clipand C-terminal SP domains, with no extra domains. Recently, C-terminal peptides from the SP domain of human thrombin, a keyenzyme in the coagulation cascade, and several S1 family memberswere reported to bind to LPS and to exert bactericidal and anti-inflammatory properties, suggesting a role for thrombin in regulat-ing the crosstalk between the coagulation system and host defensepeptides (Kasetty et al., 2011). Surprisingly, a similar C-terminalsequence pattern to that of thrombin and the S1 family (X-[PFY]-X-[AFILV]-[AFY]-[AITV]-X-[ILV]-X(5)-W-[IL]-X (Warejcka andTwining, 2005; Yount et al., 2006)), which has important functionsin the antibacterial and LPS binding activities, was also found to bepresent in the C-terminal region of PmClipSP2 (FPGVYTSVSH-YRSWVE), except for the amino acid change from I/L to V (under-lined). However, the biological activities of this region inPmClipSP2, as with human thrombin, remain to be ascertained.

In the horseshoe crab coagulation cascade, the SP zymogen, fac-tor C, acts as a PRP for microbial invaders. Factor C interacts withthe lipid A portion of LPS through the tripeptide motif (-Arg36-Trp37-Arg38-) located in the N-terminal Cys-rich region (Arikiet al., 2004; Koshiba et al., 2007). Factor G, another SP zymogenof the horseshoe crab coagulation system, acts as a sensitive andspecific sensor for b-1,3-D-glucans (Takaki et al., 2002). In theinsect M. sexta, the hemolymph proteinase HP14 is the PRP forPGN and b-1,3-glucans and is involved in triggering the proPO

activation cascade (Ji et al., 2004; Wang and Jiang, 2006). In com-parison with these SPs, PmClipSP2 is here reported to be capableof associating with LPS and b-1,3-glucan with a moderately highbinding affinity and in a saturable and dose-dependent manner.

Recent evidence suggests that proPO in the kuruma shrimp isnot only important in the immune defense, but also maintainsshrimp homeostasis (Fagutao et al., 2009). Surprisingly, withoutbacterial infection, PmClipSP2 knockdown caused a severe mortal-ity in P. monodon in the presence of either LPS or b-1,3-glucan in adose-dependent manner. This is in contrast to the five other genesin the shrimp proPO system studied (PmproPO1 and 2, PmPPAE1and 2, and PmLGBP), where a high dose injection (10 lg/g shrimp)of LPS or b-1,3-glucan into those respective knockdown shrimp didnot significantly affect their survival (Amparyup et al., 2009, 2012;Charoensapsri et al., 2009, 2011).

In addition, PmClipSP2 knockdown increased the number of via-ble bacteria found in the shrimp hemolymph in normal culture orfollowing systemic V. harveyi infection, but did not affect their sur-vival in the absence of LPS or b-1,3-glucan (data not shown). How-ever, a high mortality of PmClipSP2 knockdown shrimp wasobserved after microbial cell wall component (LPS or b-1,3-glucan)challenge, and this was not affected by the administration of anti-biotic treatment (data not shown). Thus, increasing bacteria num-bers alone in the hemolymph of PmClipSP2 knockdown shrimp isnot sufficient to cause the observed shrimp mortality.

LPS and b-glucan are known to exhibit immunostimulant activ-ities in many animals. In crustaceans, these molecules can cause asevere reduction in the circulating THC and viability of the animal(Lorenzon et al., 1999; Söderhäll et al., 2003). In support of the no-tion that the LPS- or b-1,3-glucan- induced death of PmClipSP2knockdown shrimp was mediated by the disruption of hemocytehomeostasis, is that LPS-stimulated PmClipSP2 knockdown shrimpshowed a reduced circulating THC and a significant morphologicalchange in the remaining hemocytes, such as a dysfunctional andmoribund appearance. Therefore, PmClipSP2 is not only likely tobe involved in the melanization cascade, but also to control hemo-cyte homeostasis in the presence of LPS.

PRPs represent an important first line of defense against patho-genic microorganisms. Based on their function, PRPs can be dividedinto the two groups of (i) those that signal an immune activationand (ii) those that are scavengers of pathogens and their toxic

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products. Dectin-1, a receptor for b-glucan, is a PRP for both signal-ing and scavenger receptors (Taylor et al., 2005). More recently, themannose-binding lectin of the crayfish P. leniusculus (Pl-MBL) wasshown to play an important role in the proPO system by acting as ascavenger receptor to prevent the spread of LPS in the hemolymphand so avoid further spatially widespread or temporally length-ened of the proPO activation (Wu et al., 2013). However, in con-trast to PmClipSP2, Pl-MBL is a C-type lectin that contains acarbohydrate recognition domain (CRD) in the C-terminus.

To maintain homeostasis, the animal body is equipped with apowerful system to remove circulating waste by scavenger cells,including in blood macrophages in vertebrates and hemocytes ininvertebrates. PmClipSP2 knockdown in P. monodon revealed aslight numerical (but not statistically significant) decrease in thecirculating THC and a slight change in the morphology of thehemocytes, but in the presence of LPS the PmClipSP2 knockdownshrimp, and not the control ones, showed a dramatic and signifi-cant decrease in the circulating THC and drastic morphologicalchanges, consistent with the destruction of the hemocytes. Thus,PmClipSP2 appears to play a role in the maintenance of shrimphemocyte homeostasis upon LPS challenge.

In conclusion, this is the first study to report the potentialfunction of the P. monodon clip-SP (PmClipSP2) protein as a LPSand b-glucan sensitive SP involved in the proPO activation systemand as a LPS scavenger involved in the maintenance of hemocytehomeostasis. In vivo and in vitro experiments support thatPmClipSP2 participates in the activation of the proPO system, lead-ing to melanin synthesis. Furthermore, PmClipSP2 also acts as aPRP that exhibits LPS and b-1,3-glucan binding and can act as ascavenger for LPS and thereby prevent unnecessary destructionof shrimp hemocytes. These findings increase our understandingof the mechanisms of PmClipSP2 in the regulation of melanizationand maintaining immune homeostasis caused by microbial cellwall component stimulation.

Acknowledgments

This work was supported by research grants from the ThailandResearch Fund to A.T. (TRF Senior Scholar No. RTA5580008), theThailand Research Fund and National Center for Genetic Engineer-ing and Biotechnology, National Science and Technology Develop-ment Agency to P.A. (Grant No.RSA5580046), the IntegratedInnovation Academic Center: IIAC Chulalongkorn University Cente-nary Academic Development Project and partially supported by theHigher Education Research Promotion and National Research Uni-versity Project of Thailand, Office of the Higher Education Commis-sion (CU56-FW01), Japan International Cooperation Agency (JICA),and the Visiting Fellowship under the JSPS core-University Pro-gram. W.C. is supported by a postdoctoral fellowship from theRatchadaphiseksomphot Endowment Fund, Chulalongkorn Univer-sity. K.P. is the recipient of the 90th Anniversary of ChulalongkornUniversity Fund (Ratchadaphiseksomphot Endowment Fund) andChulalongkorn University Graduate Scholarship to Commemoratethe 72nd Anniversary of His Majesty King Bhumibol Adulyadej.J.S. is the recipient of a student fellowship from the Royal GoldenJubilee Ph.D. Program, Joint Funding of the Thailand Research Fundand Chulalongkorn University. We thank Dr. Robert Douglas JohnButcher at the Publication Counseling Unit, Faculty of Science,Chulalongkorn University, for English language corrections of thismanuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.dci.2013.06.013.

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