Fish & Shellfish Immunology (2009) 26, 72e83

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Differentially expressed genes of the carpet shellclam Ruditapes decussatus against Perkinsus olseni

M. Prado-Alvarez 1, C. Gestal 1, B. Novoa, A. Figueras*

Instituto de Investigaciones Marinas, CSIC, C/Eduardo Cabello, 6, 36208 Vigo, Spain

Received 23 November 2007; revised 22 February 2008; accepted 1 March 2008Available online 18 March 2008

KEYWORDSClam;Ruditapes decussatus;Perkinsus olseni;SSH;Immune response

* Corresponding author. Tel.: þ986292762.

E-mail address: antoniofigueras@ii1 These authors contributed equally

paper.

1050-4648/$ - see front matter ª 200doi:10.1016/j.fsi.2008.03.002

Abstract SuppressioneSubtractive Hybridization (SSH) was used to identify differentially ex-pressed Ruditapes decussatus genes against the protozoan Perkinsus olseni infection. A for-ward and a reverse subtraction were carried out to identify up- and down-regulated genesin both haemocytes and gills of clams naturally infected with P. olseni. New genes, candidatesfor further investigation into the functional basis of resistance to pathogens, have beendetected for the first time in the clam (R. decussatus). A total of 305 differentially expressedsequences were obtained, 221 of them in haemocytes and 84 in gills of infected clams. Thenumber of ESTs with potential similarity with known genes was 97, 42 among them wererelated with immunity and stress related functions. The pattern of expression of the immuneselected genes was studied by quantitative PCR with samples of naturally Perkinsus infectedclams and compared with samples from an in vitro infection of clam haemocytes with Perkin-sus zoospores. The maximum expression was found 1 h post infection. The complete openreading frames of selected sequences (Rd adiponectin-C1q and Rd DAD-1) were determined.Our results provide new insights into the molecular basis of hostepathogen interactions inR. decussatus.ª 2008 Elsevier Ltd. All rights reserved.

Introduction

Parasites of the genus Perkinsus have been associated withmortalities of molluscs around the world, including oysters,clams, abalones and scallops [39]. Perkinsus olseni affects

34 986214462; fax: þ34

m.csic.es (A. Figueras).to the work presented in this

8 Elsevier Ltd. All rights reserved

the clam R. decussatus, a cultured bivalve species with im-portant commercial value in the Portuguese and Spanishcoasts, and where mortalities have been registered associ-ated with this parasite [11,12].

Although Perkinsus species have been described as seri-ous pathogens in different molluscan species, relativelylittle is known about hosteparasite interactions, and today,most of the knowledge on the clam R. decussatus immuneresponse is based on functional assays [37,46]. Bivalveslack a specific immune system and therefore they do notpossess immune memory. Molluscan defence mechanisms

.

Differential expression of genes in the Perkinsus infected carpet shell clam 73

involve cell-mediated and humoral reactions to recognizeand eliminate pathogens. The haemocytes are the cells pri-marily involved in inflammation, wound repair, encapsula-tion and phagocytosis, and it is in the hemolymph wherethe most active components of bivalves immune responseare located [40]. In clams, cellular defensive activitiesagainst Perkinsus induce hemocytic infiltration, digestivetubule atrophy, and nodule formation on gills and mantledue to inflammation [36].

Together with the traditional diagnostic methods andfunctional assays, the knowledge of defence mechanismsand specific genes expressed in clams against pathogens,and specifically against P. olseni, may improve the under-standing of the host/pathogen interaction and immuneresponse. At present, few data are available on the molec-ular basis of the immune response of bivalve molluscsagainst pathogens. Most of them are referred to Easternand Pacific oysters and the mussels [48,18,38] but very lim-ited information is available on the expressed immune genesin clams. Recently, some ESTs related to immune genes havebeen identified in R. decussatus after stimulation with a mix-ture of dead bacterial strains, including the molecularcharacterization of two new antimicrobial peptides inducedby a bacterial challenge [15]. The only study conducted ongene expression on Perkinsus infected clams has beenperformed in Korean Manila clams (R. philippinarum) usingthe ESTs from a cDNA library, and only a few transcriptsencoded by genes putatively involved in the clam immuneresponse against P. olseni have been reported [22].

The main goal of the present study is the identificationof genes involved on carpet shell clam (R. decussatus)immune response against P. olseni using the suppressionesubtractive hybridization (SSH). A comparison of immunegenes expressed in both chronic (naturally infected clams)and acute infections (in vitro infection of clam haemocyteswith P. olseni zoospores) was also conducted.

Materials and methods

Maintenance of animals, haemocyte and tissuecollection and RNA extraction

Carpet shell clams (R. decussatus) were obtained froma commercial shellfish farm. Animals were maintained inopen circuit filtered seawater tanks at 15 �C with aerationand they were fed daily with Isochrysis galbana (12� 108

cells/animal), Tetraselmis suecica (107 cells/animal) andSkeletonema costatum (3� 108 cells/animal). Prior to con-ducting the experiments, bivalves were acclimatised to ex-perimental aquaria for 1 week.

A total of 150 clams were individually analyzed for thepresence of P. olseni. To assess the level of parasite infec-tion, a piece of clam gills was stained with Lugol’s iodineand examined under a light microscope after incubation inRay’s fluid thioglycollate medium (RFTM) [41]. Simulta-neously, 1 ml of hemolymph was collected from theadductor muscle sinus of each individual, as well as a pieceof gill tissue. Haemocytes were collected after centrifuga-tion at 2500� g during 15 min at 4 �C, and both haemo-cytes and tissue samples, were kept in liquid Nitrogenuntil use.

After three days of incubation in RFTM, clam gills wereexamined using light microscopy to confirm the intensityof Perkinsus infection. Clams with very high P. olseniinfection intensity as well as uninfected ones wereselected for the experiments. Haemocyte and gill samplesof each selected individual maintained in liquid nitrogenwere combined in two pools of 20e25 clams, one withheavily infected, and one with non-infected clams. Poolswere resuspended in 6 ml of Trizol reagent (Invitrogen)each and the RNA was extracted according to the manu-facturer’s protocol.

Perkinsus zoosporulation, in vitro hemocyticstimulation and RNA extraction

To determine the immune response of R. decussatus hae-mocytes against this pathogen, in vitro P. olseni infectionwas performed, mimicking the initial acute phase of theinfection. Samples were collected at different infection-times.

Since for this assay clams have to be kept alive, Perkin-sus infected clams were identified by a nested PCR assayperformed with whole clam blood drawn from the posterioradductor muscle. The used primers were Perk-ITS S(50-CTTAGAGGAAGGAGAAGTCGTAAC-30) and Perk-ITS As(50-GCTTACTTATATGCTTAAATTCAG-30) as reported by Kotobet al. [25].

In order to obtain zoospores, the infective stage ofPerkinsus, gills of heavily infected clams were incubated inRFTM for five days in the dark at room temperature. Hypno-spores, enlarged in RFTM, were collected by centrifugationat 3000� g after digestion of the remaining tissue by incu-bation in 20 ml of 2 M NaOH per gram of wet tissue at 50 �Cfor 1 h. Then they were transferred into aerated filteredseawater with antibiotics (penicillinestreptomycin) untilmotile zoospores developed [13,16]. Live zoospores wereresuspended in filtered seawater to a concentration of 108

cells ml�1. The effect of the P. olseni zoospores on the hae-mocyte immune response was assayed by adding infectivezoospores to two pools of 10 non-infected clams (haemo-cyte: zoospore rate 1:1).

Samples were collected after 30 min 1, 3 and 24 h postinfection, by washing haemocytes in PBS, and resuspendingthem in 6 ml of Trizol reagent (Invitrogen) for RNA extrac-tion according to the manufacturer’s protocol. A negativecontrol of haemocytes without adding the P. olseni stimuluswas also collected and RNA extracted.

Suppressionesubtractive hybridization

The SuppressioneSubtractive Hybridization technique (SSH)[9] was used to characterize new genes involved in the car-pet shell clam’s innate immune response against P. olseninatural infection. Briefly, cDNA was synthesized from 1 mgof clam haemocytes or tissue RNA sample (infected andnon-infected) using the SMART PCR cDNA Synthesis Kit(Clontech). An SSH assay was then performed using thePCR-Select cDNA Subtraction Kit (Clontech) followingmanufacturer’s instruction and using the cDNA of infectedtissues as tester, and the cDNA of non-infected tissues orcontrol as driver, for forward-subtracted cDNA samples

74 M. Prado-Alvarez et al.

(genes expressed or up-regulated in infected tissuescompared with controls non-infected) and the oppositefor the reverse-subtracted cDNA samples (genes down-regulated in infected tissues compared with controlsnon-infected). These cDNAs were then used in PCR to am-plify the differentially expressed sequences. PCR mixturewas cloned using TOPO TA cloning kit (Invitrogen) and trans-formed in E. coli competent cells.

Selected colonies were amplified by PCR using NestedPCR primer 1 and 2R from the PCR-Select cDNA SubtractionKit (SSH technique). Agarose gel electrophoresis was per-formed to visualize the amplified fragments and to selectby size the samples to be sequenced. The PCR profileconsisted of: initial denaturation for 5 min at 94 �C; 35 cy-cles of 30 s denaturation at 94 �C, 30 s annealing at 65 �Cand 1.5 min elongation at 72 �C; final extension for 7 minat 72 �C. Excess of primers and nucleotides were removedby enzymatic digestion using 10 U and 1 U of ExoI andSAP, respectively (Amersham Biosciences) at 37 �C for 1 hfollowed by inactivation of the enzymes at 80 �C for15 min. DNA sequencing was performed using a BigDye ter-minator Cycle Sequencing Ready Reaction Kit and an auto-mated DNA sequencer ABI 3730.

Sequence analysis

Raw chromatograms were analyzed with Chromas 231software (Technelysium). Search for similarities with knowngenes was performed using BLAST (http://www.ncbi.nlm.nih.gov/blast/). Translation and protein analysis werecarried out using the ExPaSy tools (http://us.expasy.org/tools). Multiple sequence alignments were generated withClustal W [49]. Database search was performed using BlastXand the best annotated hit from the similarity search wasretained. Novel ESTs were deposited in GeneBank and as-signed accession numbers from EY189730 to EY189777 andfrom EY255091 to EY255098.

Identification and characterization of newimmune-related genes

In order to obtain the complete open reading frames (ORF)of selected ESTs, RACE reactions were performed usingSMART RACE cDNA Amplification Kit from Clontech accord-ing to the manufacturer’s instructions. After ligation andcloning in pGem-T Vector System (Promega) and trans-formation in Top 10F0 competent bacteria (Invitrogen),several clones were sequenced from both ends with M13

Table 1 Specific primers used for the quantitative PCR assays.

Primer name Forward prim

Adiponectin-C1q ACACAGGACGDAD-1 GCTATTGTGCIAP CAGAGGAGTTProsaposin-like TGCATTTTCTMultidomain complement related ATGCACTTGCThrombin CGTTTGTTGTClam-b-actin CGACTCTGGA

forward and reverse primers using BigDye terminator CycleSequencing Ready Reaction Kit and an automated DNAsequencer ABI 3730. The resulting sequences weresubjected to cluster analysis.

Phylogenetic analysis

Sequences of the complete ORF of DAD-1 obtained fromthe GenBank database, Mus musculus (CAA73779.1), Homosapiens (NP001335.1), Xenopus leavis (BAA03652.1), Gallusgallus (AAC60276.1), Strongylocentrotus purpuratus(XP001178079.1), Argopecten irradians (AAX56947.1),Araneus ventricosus (AAN86571.1), Oryza sativa(BAA24072.1), Saccharomyces cerevisiae (AAC49086.1),together with the sequence obtained in this study werealigned with Clustal W [49] including in MEGA 3 [26]. ForC1q the same process was conducted with the followingcomplete ORF sequences obtained from GenBank: C1q AH. sapiens (P02745), C1q B H. sapiens (AAV38388.1), Sialicacid binding lectin Cepaea hortensis (Q70SH0), C1q Xenopuslaevis (MGC80872), Adiponectin-C1q M. musculus (Q6GTX4),C1q Cyprinus carpio (Q6L7J6), Sialic acid binding lectinHelix pomatia (Q1KM18), C1q C Homo. sapiens (P02747),Adiponectin Danio rerio (XP691074), C1q-adiponectinGallus. gallus (NP996874.1), Adiponectin Oryctolaguscuniculus (Q2LDA4), HM binding protein Mytilus edulis(Q6UQ16), C1q Lethenteron Japonicum (BAD22833.1),Adiponectin Anas Platyrhynchos (ABE03631.1).

A phylogenetic tree based on deduced amino acid ORFsequences was performed using the Neighbour-Joining (NJ)algorithm with the MEGA 3 software programme. Statisticalconfidence on the inferred phylogenetic relationships wasassessed by bootstrap of 10.000 replicates.

Expression analysis of the identified immune genes

To determine the expression of several selected up-regulated ESTs from the SSH library and to evaluate andquantify their relative expression, a real time SYBR GreenPCR assay was carried out with the total haemocytes cDNAof both natural conditions and also in vitro P. olseniinfections at different times.

Quantitative PCR assays and data analysis were per-formed using a 7300 Real Time PCR System (AppliedBiosystems). The 25 ml PCR mixture include of 12.5 ml ofSYBR Green PCR master mix (Applied Biosystems) with0.5 ml of primers pairs 10 mM designed for the selectedsequences [42] (Table 1), and 1 ml of a 1:5 dilution of the

er Reverse primer

TCTCCATTCC AGTTCTTTTCTTCGGCAGCAATTGGTTGGA AATGCTCTTTCTGGGCTGATGCAGTCGGTA TACATCTGCCCTTTTGTCCA

TTTGCTTTCG TGTTTGGTAGCCCCACATTAGAAAGATCG AAATGCAACATCCAACAGGATCGACATCCT ATGATCCTTGTTCCGCTTTCGATGGTGTCA ATGAGTAAGTGTTGGTGGCG

Differential expression of genes in the Perkinsus infected carpet shell clam 75

cDNA. Amplification was carried out at the standard cyclingconditions of 95 �C for 10 min, followed by 40 cycles of95 �C 15 s and 60 �C for 1 min. The comparative CT method(2-DDCT method) was used to determine the expressionlevel of analyzed genes [29]. The expression of the candi-date genes was normalized using b-actin as a housekeepinggene (Table 1). Fold units were calculated dividing the nor-malized expression values of infected tissues by the nor-malized expression values of the controls. Results aregiven as the mean and standard deviation of two replicatesand pools.

Results

ESTs sequencing

A total of 305 clones (142 haemocyte-P. olseni up-regu-lated, 79 haemocyte-P. olseni down-regulated, 49 gill-P. ol-seni up-regulated and 35 gill-P. olseni down-regulated)obtained from forward and reverse SSH libraries were iso-lated, amplified by PCR, sequenced and analyzed. BLASTXanalysis and comparison of ESTs against GenBank databaseswere performed to find sequence similarity.

The detected genes were clustered and graphicallyrepresented into four functional categories (Fig. 1). Ahigh number of the transcripts in the forward libraries(20%) were potentially involved in immune response andstress: 37 ESTs in haemocytes and 1 in gills. However, lessimmune and stress related genes were detected in the

No hit62%

Unknown function6%

Cell processes12%

Immunity/stress20%

No hit60%

Immunity/stress

4%Cell processes28%

Unknown function8%

A

B

Figure 1 A. Functional classification of ESTs correspondingto genes identified as up-regulated in R. decussatus infectedby P. olseni. The ESTs were clustered into four categories ac-cording to their putative biological function. B. Functionalclassification of ESTs corresponding to genes identified asdown-regulated in R. decussatus infected by P. olseni. TheESTs were clustered into four categories according to their pu-tative biological function.

two reverse libraries (4%). Concerning ESTs potentially in-volved in cell processes, a total of 23 ESTs (12%) and 32ESTs (28%) were found in the forward and reverse libraries,respectively. The remaining 118 transcripts (62%) corre-sponded to up-regulated genes and 69 transcripts (60%) cor-responded to down-regulated genes from haemocytes andgills of P. olseni infected clams had no significant hits onGenBank.

An overview of the obtained results is displayed inTable 2. Only matches with e-values smaller than 10e�3

were retained as significant, giving a total of 56 ESTsgrouped in 39 contigs. A detailed characterization of theputative function of the ESTs with significant match ob-tained in the forward and reverse libraries are shown inTables 3A and 3B, respectively.

A total of 28 sequences obtained from the forward SSHlibrary of haemocytes grouped in 20 unique consensussequences were potentially protein-coding genes, amongthem 17 ESTs grouped in eight unique consensus sequences,found in the haemocytes forward library, were putativelyrelated with immunity and stress. Five ESTs showedsimilarity with C1q-adiponectin, three ESTs showed simi-larity with a multidomain that included a sushi, a VonWillebrand factor type A, an EGF and a pentraxin domainand two ESTs had a similarity with the serine protease,thrombin. Two sequences had high homology with DAD(Defender Against Death), protein possibly related withinjury and apoptosis. Three transcripts shared a saposindomain, among them two showed similarity with the poly-peptide prosaposin-like, and one with saposin-PA isoform A.A transcript with a similarity to the Zinc dependent metal-loendopeptidase astacin, was also found. One overex-pressed EST had similarity to chaperonin, a stress relatedprotein. In addition, a serine protease with a similarity toa salivary protein was identified as up-regulated in gills ofinfected clams. In the reverse libraries, identified as down-regulated in haemocytes of clams infected by Perkinsus,only four ESTs were related with immunity and stress,a transcript matched ferritin and three transcripts witha similarity to IAP (inhibition of apoptosis protein).

A total of 10 ESTs in the forward libraries were putativelyinvolved in cell processes and only one of them was foundas overexpressed in the gill. However, most of the down-regulated ESTs (23 of 27) were putatively related withcell process functions: 15 of them found in haemocytesgrouped in 12 unique consensus sequences and implicatedin cell structure, metabolic processes and cell signaling

Table 2 General characteristic of ESTS obtained fromhaemocytes of R. decussatus naturally infected byPerkinsus olseni.

Category Total ESTs

Total number of EST sequenced 404Total number of EST analyzed 305Significant matches to known proteins 56EST clusters 39Singletons 26No significant match to database 187Redundancy 69%

Table 3A Identified SSH up-regulated clones in chronic P. olseni infected clams R. decussatus with significant database match.

Category and gene identity. BlastX Genbankaccession no.

Insertsize (bp)

Homolog species e-Value no. sec./no. contig

Immunity and stressAdiponectin-C1q (H) EY255091 576 Anas platyrhynchos 3e�07 5/1

EY255092 576 A. platyrhynchos 4e�07EY189761 656 A. platyrhynchos 8e�07EY255093 576 A. platyrhynchos 9e�07EY189744 640 A. platyrhynchos 8e�07

DAD-1 (H) EY189760 337 Apis mellifera 1e�40 2/1EY255094 303 A. mellifera 2e�39

Thrombin (H) EY189742 809 Gekko gecko 3e�11 2/1EY189743 844 G. gecko 9e�11

Sushi, von Willebrand factor type A, EGF,pentraxin (multidomain complementrelated) (H)

EY189748 647 Bos taurus 1e�3 3/1EY189749 646 B. Taurus 1e�3EY189750 647 B. taurus 1e�3

Saposin related CG12070-PA isoform A (H) EY189747 270 D. melanogaster 5e�10 1/1

Naegleriapore A pore-forming peptide precursor(Prosaposin-like) (H)

EY189740 743 Naegleria fowleri 3e�11 2/1EY189741 753 N. fowleri 3e�11

Astacin (metalloendopeptidase FARM-1) (H) EY189746 519 Hydra vulgaris 1e�25 1/1

Putative salivary protein (serine protease) (G) EY189731 214 Culicoides sonorensis 6e�09 1/1Cct5-prov protein (Chaperonin) (H) EY189745 634 Xenopus laevis 1e�88 1/1Cell processCell structureActin (H) EY189751 454 Bionpharia glabrata 2e�75 1/1Similar to protein UNQ6350/PRO21055 homolog

precursor (H)EY189756 428 Canis familiaris 6e�07 1/1

Fertilization envelope outer layer protein (H) EY189759 426 Siniperca chuatsi 2e�06 1/1

Metabolic processCytochrome oxidase 1 (H) EY189752 790 R decussatus 6e�72 1/1Betaine homocysteine methyl transferase (H) EY189753 255 S. purpuratus 5e�42 1/1Alpha-amylase (H) EY189754 303 Crassostrea gigas 2e�37 1/1Similar to MGC82104 protein (H) EY189755 255 Danio rerio 2e�15 1/1Methylmalonate semialdehyde

dehydrogenase-like protein (H)EY189757 351 A. elegantissima 5e�50 1/1

Signal peptide protease CG11840-PA (H) EY189758 335 D. melanogaster 9e�48 1/1Cytochrome c subunit II (G) EY189730 280 Ruditapes philippinarum 5e�11 1/1

Unknown functionC09D4.2 (H) EY255095 529 Caenorhabditis elegans 4e�4 1/1CG9246-PA (H) EY255096 249 Gallus gallus 2e�22 1/1

Clones found in the haemocyte library are indicated with (H) and those found in the gill library with (G).

76 M. Prado-Alvarez et al.

functions and eight of them, grouped into three uniqueconsensus sequences, were putatively related with meta-bolic process and belong to gill.

Characterization of new immune-related genes

Once some ESTs with putative immune function weredetected, two of them, a C1q-adiponectin A like proteinand a defender against death 1 (DAD) like protein, wereselected by their ESTs frequency and their possible impli-cation on the defense against Perkinsus.

A total of five sequences forming a cluster wereputatively identified as homologous to a complement

pathway protein C1q-adiponectin. The 50 RACE gave asa result a new protein of 633 bp ORF encoding 211 aminoacidic residues. A signal peptide of 23 amino acids was lo-calized in the N-terminal region showing a cleavage sitestarting after the S preceding the A, at position 120 ofthe amino acid sequence. A complete C1q domain contain-ing 130 amino acid residues was identified in the ORF se-quence (Fig. 2A). The new protein was named Rdadiponectin-C1q.

The phylogenetic tree constructed with the deduced Rdadiponectin-C1q amino acid sequence (Fig. 2B) and withselected vertebrate and invertebrate sequences containingthe C1q domain showed two clusters, one corresponding to

Table 3B Identified SSH down-regulated clones in chronic P. olseni infected clams R. decussatus with significant databasematch.

Category and gene identity. BlastX GenbankAccession no.

Insertsize (bp)

Homolog species e-Value no. sec./no. contig

Immunity and stressIAP repeat-containing protein 2

isoform (H)EY189762 593 Pan troglodytes 2e�12 2/1EY189763 587 P. troglodytes 2e�12

IAP repeat-containing protein 6 (H) EY189773 253 Danio rerio 1e�10 1/1Yolk ferritin (H) EY189764 320 Paragonimus westermani 3e�06 1/1Cell processCell structureGTP binding protein 4 (H) EY189765 605 Gallus gallus 2e�89 1/1

Actin (H) EY189766 391 Biomphalaria glabrata 3e�57 2/1EY189767 397 B. glabrata 3e�57EY255097 300 Drosophila melanogaster 4e�21 1/1EY255098 382 Hypophthalmichthys molitrix 2e�36 1/1

Elongation factor 1 beta (H) EY189769 369 Plutella xylostella 2e�35 1/1

NADH-ubiquinone oxidoreductase (H) EY189775 502 Anopheles funestus 4e�43 1/1

Metabolic processMitochondrial Hþ ATPase a subunit (H) EY189768 297 Pinctada fucata 2e�41 1/1Cytochrome c oxidase subunit III (H) EY189770 498 Acanthocardia tuberculata 5e�27 2/1

EY189771 858 A. tuberculata 5e�27Apg3 p (autophagocytosis associated

protein) (H)EY189772 370 Macaca mulatta 5e�14 1/1

Thyroglobulin precursor (H) EY189776 515 Tribolium castaneum 5e�06 2/1EY189777 521 Aedes aegypti 4e�08

Cytochrome oxidase subunit 1 (G) EY189732 780 R. philippinarum 1e�72 5/2EY189733 676 R. philippinarum 1e�63EY189736 732 R. philippinarum 1e�11EY189738 638 R. philippinarum 2e�20EY189739 526 R. philippinarum 4e�12

Cytochrome c oxidase subunit III (G) EY189734 400 A. tuberculata 4e�27 2/1EY189735 733 R. philippinarum 3e�36

NADH dehydrogenase subunit 5 (G) EY189737 588 R. philippinarum 4e�09 1/1Cell signalling/communicationChain B, Cdc42 complexed with

the Gtpase binding domain of P21activated kinase (H)

EY189774 322 Rattus norvegicus 6e�05 1/1

Clones found in the haemocyte library are indicated with (H) and those found in the gill library with (G).

Differential expression of genes in the Perkinsus infected carpet shell clam 77

vertebrate sequences (mammals, birds and fish) and a sec-ond one grouping the invertebrates.

The sequence clustering and BLASTx similarity searchingof the SSH obtained ESTs showed two sequences forminga cluster that can be putatively identified as homologous toa defender against death 1 protein in R. decussatus. Afteramplification of the complete ORF by 50 RACE, cloningand sequencing, the full length ORF nucleotide and aminoacid sequences were obtained (Fig. 3A). The new DAD-1gene identified in clams (Rd DAD-1) presented a 339 bpORF encoding 113 amino acid residues. A signal peptidewas localized in the N-terminal region. A complete DADdomain was identified in the ORF sequence, and includesthree transmembrane helices, indicating that clam-DAD-1is an integral membrane protein (Fig. 3A).

A multiple sequence alignment of the deduced clam-DAD-1 amino acid sequence with those described in othermarine invertebrate species revealed a relative similarity,differing in 31 amino acid residues (Fig. 3B), and showingbetween 73% and 75% of nucleotide homology with the pre-viously described DAD-1 genes.

The phylogenetic tree constructed with the amino acidsequences of the herein described clam Rd DAD-1, togetherwith different described DAD-1 proteins in both animalsand plants showed two clusters, one corresponding toanimal DAD-1, and the other corresponding to plant DAD-1 [23]. The yeast S. cerevisiae was used as outgroup.Neighbour-Joining phylogenetic analysis supported RdDAD-1 as a separate taxon into the DAD-1 animal cluster(Fig. 3C).

1 ccgtctcttttatcggaaggtgttgaagcaattagcacagaaataatacagaaaaaaaac 60

61 cacttcagaatgatgatgatattttgcttcgtgttagttttatcgcaaggaacgctaagt 120 M M M I F C F V L V L S Q G T L S

121 gcgtcacttccggagagtagcgatacaagcaagaaccttgagtcgtttgtacagaatttg 180 A S L P E S S D T S K N L E S F V Q N L181 tttacgggaagcagtgatctatcaaatgagttcagttttgaggaaaccgaggatgaaatt 240 F T G S S D L S N E F S F E E T E D E I241 ccaccacatgaagaagaaacacaggacgtctccattcctgagaacggacttttatttaaa 300 P P H E E E T Q D V S I P E N G L L F K301 caaggatttcatgcctacctcagcggtccacggtgctaccacacaggcaacccaattaaa 360 Q G F H A Y L S G P R C Y H T G N P I K361 tttgacgtggcatcggtaaatattggtaacagatacagtgtccatactggtgtatatgtg 420 F D V A S V N I G N R Y S V H T G V Y V421 gtggccacacctggactctacttttttacctggaccgttgctgccgaagaaaagaactgg 480 V A T P G L Y F F T W T V A A E E K N W481 tttgtgtctgatttaatggtgaacggggttctgagaggaaacaccatgaccgactcatat 540 F V S D L M V N G V L R G N T M T D S Y541 ggaaatggaaaagggaacggcatccatccagcgaccggatttgtcttggtaaatgtcaaa 600 G N G K G N G I H P A T G F V L V N V K601 cgcggggatcatgtttacataagatttgtcaccggtcatggatgcattataaaaagcaac 660 R G D H V Y I R F V T G H G C I I K S N661 aggatgactcgatctacgttttctggatggcgtttgcactaagcaacttgggcgacagtc 720 R M T R S T F S G W R L H *721 tactgatgctggaaaaaaggagaagcttatcgacacaaccgttaaaccattaattcttgc 780

781 cgaatctcacacgttcttcagtttattcgtttctaacattttttctttatttttatttct 840

841 aaaatacgtacctcg

A

B

Figure 2 A. Complete ORF nucleotide and deduced amino acid sequences of the clam C1q. (*) Indicates the stop codon. C1qdomain is in grey. (;) Indicates the cleavage site of the signal peptide. Underlying indicates transmembrane peptide. B. Neigh-bour-Joining (NJ) tree of the ORF amino acid sequence data shows the phylogenetic relationships among the new clam adiponec-tin-C1q and the complete ORF of previously reported proteins containing C1q domain in other species. Bootstrap: 10 000repetitions.

78 M. Prado-Alvarez et al.

Expression patterns of the identifiedimmune-related genes

Quantitative PCR analysis was performed to determine therelative expression pattern of several immune-relatedgenes identified by SSH as up-regulated in haemocytesfrom naturally P. olseni infected clams. A significant

induction of the expression levels of the selected immune-related genes (Rd adiponectin-C1q, Rd DAD-1, Prosaposin-like, multidomain complement related protein and throm-bin) were detected in infected clams in contrast with non-infected or controls (Fig. 4A). Chronic Perkinsus infectioninduced an increase of 30.6; 9.11; 3.64; 12.34; and 4-foldsrespectively.

1 cagcggagaaccagcgatcggtgtctacttccgggaaattgccacgtccaatttttctca 60

61 cttgtttgcaagaaatagcattatttaacaatgccagagaaactaacgggagtcatattc 120M P E K L T G V I F

121 aagttttatgacgagtacataaacagtactcctaagagactcaaaattatagacgcatac 180 K F Y D E Y I N S T P K R L K I I D A Y

181 ctgctctatattttcctgacaggtgtatttcagttttgctattgtgcattggttggaact 240 L L Y I F L T G V F Q F C Y C A L V G T

241 ttcccattcaattccttcctgtcagggttcatctcatgtgtaggatcttttgttattgca 300 F P F N S F L S G F I S C V G S F V I A

301 gtatgtttaagattacaagtaaatccacagaataagtcagacttttccggcatcagccca 360 V C L R L Q V N P Q N K S D F S G I S P

361 gaaagagcatttgctgattttatcttcgcaaatttggttctacacttggtggttatgaac 420 E R A F A D F I F A N L V L H L V V M N

421 ttcattggttaaatggaccatcttgttggagta 453 F I G *

R. decussatus MPEKLTGVIFKFYDEYINSTPKRLKIIDAYLLYIFLTGVFQFCYCALVGTFPFNSFLSGFISCVGSFVIAA. irradians MPDSLFSVVKKFTDEYISSTKPKLKIVDAYLFYILLTGVIQFMYCALVGTFPFNSFLSGFISSVGSFVLGS. purpuratus MSVNLFTVINKFYDEYTTRTPQKLKIIDAYLTYILLTGIVQFVYCALVGTFPFNSFLSGFISSVGSFVLA *. .* *: ** *** . **::***:**** **:***:.** *******************.*****:.

R. decussatus VCLRLQVNPQNKSDFSGISPERAFADFIFANLVLHLVVMNFIGA. irradians VCLRLQVNPQNKHDFTGIGPERAFADFIFAHIILHLVVINFIGS. purpuratus VCLRLQVNPANKSSFQGISSERAFADFIFASVILHLVVMNFIG ********* ** .* **..********** ::*****:****

DAD-1 Mus musculus

DAD-1 H. sapiens

DAD-1 Xenopus leavis

DAD-1 Gallus gallus

DAD1 S. purpuratus

DAD1 Argopecten irradians

DAD1 Ruditapes decussatus

DD-1 Araneus ventricosus

DAD-1 Oryza sativa

oligosaccharyltransf. Saccharomyces cere

9559

65

66

35

1824

0.2

A

B

C

Figure 3 A. Complete ORF nucleotide and deduced amino acid sequences of the clam-DAD-1. (*) Indicates the stop codon. (;)Indicates the cleavage site of the signal peptide. DAD domain is in grey. The three transmembrane helices are underlined. B. Mul-tiple alignment of clam-DAD-1 compared with those described in other invertebrate species. (*) identical residues, (:) conservedsubstitutions and (.) semiconserved substitutions. The most similar sequences are highlighted in grey, and the aminoacidic differ-ences between these two forms are in bold. C. Neighbour-Joining (NJ) tree of the ORF amino acid sequence data shows the phy-logenetic relationships among the new clam-DAD-1 and the complete ORF of DAD-1 previously reported in other species. Bootstrap:10 000 repetitions.

Differential expression of genes in the Perkinsus infected carpet shell clam 79

Temporal expression analysis of the selected immune-related genes was also measured by quantitative PCR on invitro P. olseni infected haemocytes. Fig. 4B shows the ex-pression level of infected samples compared with controls.The expression of immune-related genes reached maximumlevels at 1 h post infection (6-folds for Rd adiponectin-C1q;2.6-folds for Rd DAD-1; 2.24-folds for prosaposin-like; 2-5folds for multidomain related protein and 4.2-folds forthrombin). After 1 h post infection, the expression levelof these selected genes decreased to the control.

Discussion

Infectious diseases remain a major concern for bivalveaquaculture and may result in massive losses. Management

of infectious diseases including the understanding of themolecular mechanisms and genes implicated in the hosteparasite relationships and immune response is a priorityfor aquaculture sustainability and progress. However, de-spite some specific findings, little is known concerning themolecular mechanisms involved in the recognition, activa-tion and effector molecules of bivalve immune response topathogens [15,18,22,38,48]. Some information is availableon the response of bivalves against contaminants andstress conditions [3,7,10,20,44,47,50]. Furthermore, mostof the studies of genes involved in the immune responsehave been performed in oysters, and little is known aboutthe molecular immune response in other bivalves againstpathogens, including those produced by clams againstpathogens [15,22]. The major goal of the present studywas to determine the ESTs with immune role. However,

0

5

10

15

20

25

30

35

Rd Adiponectin-C1q

Rd DAD-1 Prosaposin like

Multidomaincomplement

related

Thrombin

Rd Adiponectin-C1q

Rd DAD-1 Prosaposin like

Multidomaincomplement

related

Thrombin

Ex

pre

ss

io

n le

ve

l

*

*

*

*

*

01234567

Ex

pre

ss

io

n le

ve

l

30´ 1h 3h 24h

*

A

B

Figure 4 A. Quantitative expression of immune-relatedgenes identified as up-regulated by SSH in clams R. decussatushaemocytes, with chronic infection by P. olseni in naturalconditions. Results are mean� SD. Bars represent the relativeexpression transcript levels of infected clams referred tocontrols, previously normalized to b-actin transcript levels.Asterisk * indicates significant differences with controlsp< 0.05. B. Quantitative expression of immune-related genesexpressed after in vitro infection of clam R. decussatus haemo-cytes by zoospores of P. olseni, analyzed at 30 min, 1, 3 and24 h post infection. Results are mean� SD. Bars representthe relative expression transcript levels of infected clamsreferred to controls, previously normalized to b-actin tran-script levels. Asterisk * indicates significant differences withcontrols p< 0.05.

80 M. Prado-Alvarez et al.

all hits were examined for their potential role in thedisease process.

The suppressionesubtractive hybridization method (SSH)is a PCR-based technique that allows the identification ofgenes differentially expressed in response to stimuli,combining normalization and subtraction. This method iscurrently being used to identify genes involved in differentmolecular mechanisms not only in aquatic organisms, butalso in many other different taxa. Interestingly, in the pres-ent study, most of the ESTs with putative immune functionwere detected in haemocytes supporting that these cellsplay an important role in the defense against pathogens.This had been previously supported by functional studiesand more evidence is being gathered on the molecular as-pects. Moreover, P. olseni is an intracellular parasite whichcan be found parasitizing, among other cell types,

haemocytes and this may have some influence in the mod-ulation of the gene expression.

In addition, and even though the results obtained by SSHcan be considered successful, it is known that bivalvegenomics is at its beginning, and the available data refersonly to a very small number of genes [43,50], which compli-cates the analysis of similarity, annotation and functionalclassification of new sequences. Some of the genes identi-fied by similarity search through the GenBank databasewere not previously characterized in bivalves. Most of thesequences characterized in the present work are newESTs, and some of them have unknown functions in clams.Further studies are needed to characterize those genes,which will be the goal of future researches.

The identification of transcripts showed in the SSHmatching to proteins involved in complement signalingsuch as Rd adiponectin-C1q and a multidomain proteinwith a similarity to complement related domains. Amongthem, we identified a protein similar to Thrombin, a serineprotease involved in coagulation and stimulation of cellularresponses, implicated in inflammation process and tissuerepairing in vertebrates [2]. Three transcripts with a homol-ogy to a saposin domain and similarity to Prosaposin andSaposin A were also identified. Prosaposin is a glycoproteinthat is proteolytically processed to saposin, exists in lyso-somes and also as an integral membrane protein [24]. Sapo-sin has been associated to lytic processes, pore-formationand membrane binding [17,19]. In the present study, thesegenes are overexpressed in Perkinsus infected haemocytes.Indeed, the increased expression level of those immune-related genes in response to Perkinsus infection suggeststhat these molecules could be key factors in the immuneresponse against pathogens.

A complement system with opsonic and lytic effectorpathways had been thought to exist exclusively in verte-brates. However, the identification of vertebrate C3 homo-logs with opsonic activity and potential C3 convertases insea urchins and tunicates suggests an earlier developmentof the complement system in the lower deuterostomes[31,34,35]. Interestingly, most domains found in the mam-malian complement components, except for MAC/Perforin(MACP), C1q, and factor I Module (FIM) domains, seem tohave an early origin in the metazoa lineage [34]. However,in the present study we have identified several ESTs witha C1q domain in the clam, overexpressed after infectionthat could be related with the lectin complement pathwayin the innate immunity which is closely related to the clas-sical complement pathway in adaptive immunity. The factthat the clam Adiponectin-C1q clusters together in thephylogenetic tree with the Sialic Acid binding lectin mightbe in line with the hypothesis that the classical pathwayemerged after the lectin pathway, and that the activationmechanism of the latter was partially conserved. In fact,similarities between these two pathways of complementactivation can be found in the structure and function oftheir components. Both pathways are initiated by com-plexes consisting of collagenous proteins and serine prote-ases of the mannose-binding lectin (MBL)-associated serineprotease (MASP)/C1r/C1s family.

The classical and lectin pathways can be traced back toat least cartilaginous fish [32] and ascidian (urochordata)[1,21,30,52,] respectively. The two C1q-like genes

Differential expression of genes in the Perkinsus infected carpet shell clam 81

described in Ciona intestinalis genome [8,30] have a colla-gen and C1q domains closely related to human CQT 1 [C1qand tumor necrosis factor (TNF)-related proteins] andCQT3, respectively, belonging to the C1q/TNF superfamily[34] but is not evident if it will play a similar role to verte-brate C1q as it may happen in the presently reported Rdadiponectin-C1q-like. Further investigations are needed toelucidate this aim, and will be the objective of a separatepublication.

Apoptosis or programmed cell death (that essentiallyacts on the elimination of unwanted cells [5,14]) can playan important role in the progression of disease, and nor-mally occurs when a cell is damaged, infected or senes-cent [45] and also plays an important role during theelimination of harmful cells [14]. The DAD-1 (defenderagainst apoptotic cell death) is involved, among otherprocesses, in the genetic control of apoptosis inhibitingit [33], and it is known to be a highly conserved geneamong humans, mice, clawed frogs, plants, and even re-cently have been characterized in arachnids, and in themarine invertebrate A. irradians [53] and S. purpuratus(GenBank accession number XP001178079) suggestingthat this gene could have similar biochemical functionsin animal and plants. The DAD expression could also berelated with damage caused by the infection like analteration of the normal state. In fact, DAD expressionwas inducted by bacterial infection [28], after thermalshock in the spider A. ventricosus [27] and even injurystimuli [53] leading to the idea of over expression afterundesirable stimulus [27]. Little is known on the effectand role of DAD-1 as inhibitor of programmed cell deathin molluscs. Branchial lesions associated with apoptoticcells that could be related to temperature changes andseasonality have been described in oysters O. edulis [6].In addition, Sunila and LaBanca [45] showed that oysterCrassostrea virginica infected with P. olseni presenteda reduced number of apoptotic cells in contrast witha large number of apoptotic haemocytes observed innon-infected oysters, suggesting that the parasite mayprevent haemocyte apoptosis in order to guarantee theirsurviving inside the host. In consequence, additional stud-ies are needed to ascertain the real action mechanism ofthis gene in bivalve molluscs.

The relative expression level of the selected asputatively immune-related genes showed a similar pat-tern in both natural chronic and in vitro infection. How-ever, even though the higher expression level ofimmune-related genes is produced in vitro at 1 h post in-fection, and dropped at next 3 and 24 h, the chronic ex-pression level of most of the selected immune genesindicated that they are probably expressed and activesduring the course of the infection. Chintala et al. [4]indicated that curiously the comparison between chronicversus acute infections of C. virginica by P. olseni showedthat the parasite loads stabilized over time at approxi-mately the same levels in both treatments. As in ourstudy, the decrease in immune-related expression levelsin C. virginica after the first hour could indicate thatthe host initially controls the infection, starting a processof elimination of the parasite [4]. However, it is knownthat Perkinsus infection in natural conditions is a continu-ous reinfection [51] which probably makes necessary to

maintain a continuous high expression level of specificimmune genes.

To our knowledge, the present work constitutes the firstanalysis on R. decussatus immune-related genes againstP. olseni infection using the suppressionesubtractive hy-bridization technique. Further study of the described genesis needed since the identification of molecular markersrelated to P. olseni resistance could be quite useful forselection purposes in bivalve aquaculture.

Acknowledgements

This work has been partially funded by the EuropeanCommission through the Marie Curie European Reintegra-tion Project ERG-CT-2005-518007 and the Spanish MEC(AGL2003-02454). Maria Prado wishes to acknowledge CSICfor her I3P fellowship. Camino Gestal wishes to acknowl-edge additional funding from the Spanish Ministerio deEducacion y Ciencia through the ‘‘Ramon y Cajal’’Contract.

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