Differential expression of genes encoding anti-oxidant enzymes in Sydney rock oysters, Saccostrea glomerata (Gould) selected for disease resistance

Fish & Shellfish Immunology 26 (2009) 799–810

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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi

Differential expression of genes encoding anti-oxidant enzymes in Sydney rock oysters, Saccostrea glomerata (Gould) selected for disease resistance Timothy J. Green a, *, Tom J. Dixon b, c, Emilie Devic a, Robert D. Adlard d, e, Andrew C. Barnes a, f a

The University of Queensland, Centre for Marine Studies, Brisbane, Queensland 4072, Australia CSIRO Livestock Industries, QLD Biosciences Precinct, St Lucia 4072, Australia c CSIRO Food Futures National Research Flagship, 5 Julius Avenue, North Ryde, NSW 2113, Australia d Biodiversity Program, Queensland Museum, South Bank 4101, Australia e The University of Queensland, School of Molecular & Microbial Sciences, Brisbane, Queensland 4072, Australia f The University of Queensland, School of Biological Sciences, Queensland 4072, Australia b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 January 2009 Received in revised form 17 March 2009 Accepted 17 March 2009 Available online 28 March 2009

Sydney rock oysters (Saccostrea glomerata) selectively bred for disease resistance (R) and wild-caught control oysters (W) were exposed to a field infection of disseminating neoplasia. Cumulative mortality of W oysters (31.7%) was significantly greater than R oysters (0.0%) over the 118 days of the experiment. In an attempt to understand the biochemical and molecular pathways involved in disease resistance, differentially expressed sequence tags (ESTs) between R and W S. glomerata hemocytes were identified using the PCR technique, suppression subtractive hybridisation (SSH). Sequencing of 300 clones from two SSH libraries revealed 183 distinct sequences of which 113 shared high similarity to sequences in the public databases. Putative function could be assigned to 64 of the sequences. Expression of nine ESTs homologous to genes previously shown to be involved in bivalve immunity was further studied using quantitative reverse-transcriptase PCR (qRT-PCR). The base-line expression of an extracellular superoxide dismutase (ecSOD) and a small heat shock protein (sHsP) were significantly increased, whilst peroxiredoxin 6 (Prx6) and interferon inhibiting cytokine factor (IK) were significantly decreased in R oysters. From these results it was hypothesised that R oysters would be able to generate the anti-parasitic compound, hydrogen peroxide (H2O2) faster and to higher concentrations during respiratory burst due to the differential expression of genes for the two anti-oxidant enzymes of ecSOD and Prx6. To investigate this hypothesis, protein extracts from hemolymph were analysed for oxidative burst enzyme activity. Analysis of the cell free hemolymph proteins separated by native-polyacrylamide gel electrophoresis (PAGE) failed to detect true superoxide dismutase (SOD) activity by assaying dismutation of superoxide anion in zymograms. However, the ecSOD enzyme appears to generate hydrogen peroxide, presumably via another process, which is yet to be elucidated. This corroborates our hypothesis, whilst phylogenetic analysis of the complete coding sequence (CDS) of the S. glomerata ecSOD gene is supportive of the atypical nature of the ecSOD enzyme. Results obtained from this work further the current understanding of the molecular mechanisms involved in resistance to disease in this economically important bivalve, and shed further light on the anomalous oxidative processes involved. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Oyster Saccostrea Neoplasia Innate immunity Gene expression Superoxide dismutase Peroxiredoxin

1. Introduction Oyster farming is unique with regards to disease management as there are very few ways to reduce the detrimental impact of pathogens on oyster production. Oysters are grown in estuaries, which strongly preclude the potential use of chemotherapy due to the quantity of product required, and the impact on the environment.

* Corresponding author. Tel.: þ61 7 33467289; fax: þ61 7 33654755. E-mail address: [email protected] (T.J. Green). 1050-4648/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.03.003

There is also a high likelihood of re-infection [1]. Consequently, control of diseases in oyster production currently relies on disease management practices, and in the long-term successful introduction of disease resistant stock [1]. The Sydney rock oyster, Saccostrea glomerata (previously Saccostrea commercialis), is a commercially important aquaculture species in New South Wales and South East Queensland, Australia. Since the 1970’s, mass mortalities of farmed S. glomerata have been observed due to QX disease (caused by a paramyxean protozoan, Marteilia sydneyi) and Winter Mortality (caused by a haplosporidian protozoan, Bonamia roughleyi) [2]. The current method of farming

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T.J. Green et al. / Fish & Shellfish Immunology 26 (2009) 799–810

S. glomerata is by catching wild spat on tar-coated sticks in estuaries with high spat fall [2]. Juvenile oysters are then transferred to estuaries known for having good growing characteristics for the rest of the production cycle. This method does not allow for genetic improvement within the stock for increased disease resistance. In 1996, New South Wales Department of Primary Industries (NSW DPI) initiated a hatchery based disease resistance breeding program by inbreeding offspring from survivors of field outbreaks of the protozoan parasitic diseases of QX disease and Winter Mortality over several generations [3]. Although this has been an effective program with increased resistance to these two diseases reported after several generations [3], the technique has a number of disadvantages including: A decline in overall fitness of the stock due to inbreeding, fluctuating disease intensity between years, and the possibility of selection of survivors based on environmental rather than genetic factors [4,5]. Thus, identification of genetic markers for disease resistance in S. glomerata would be useful in enhancing the current S. glomerata selection program by allowing marker assisted selection (MAS) of brood-stock without the need for prior disease challenge. Secondly, development of markers for disease resistance would allow genetic improvements within the stock between different generations to be accurately quantified by genotyping. To date, work on identifying markers for disease resistance in S. glomerata has focused on QX disease and the enzyme phenoloxidase using proteomics. Peters and Raftos [6] reported that QX infection lowers the level of phenoloxidase activity in the hemolymph of S. glomerata. Subsequent work has shown that S. glomerata bred for QX resistance have higher phenoloxidase activity in their hemolymph than unselected controls [7] and a higher proportion of oysters bred for QX resistance contain a novel form of the phenoloxidase enzyme as observed by native-polyacrylamide gel electrophoresis [5,8]. However, neither the underlying genetic basis for these polymorphisms in the phenoloxidase enzyme, nor the biochemical advantage of expression of the novel phenoloxidase phenotype has been reported. In the current study, a line of oysters bred for resistance to QX disease (R) [9] was compared to unselected control oysters (W) by culturing both simultaneously on an intertidal commercial oyster lease in the Pimpama River, SE Queensland, Australia. Although there was no indication of disease outbreaks caused by QX disease or Winter Mortality, significant mortality was observed only in the W oysters resulting from another disease identified as disseminating neoplasia [10]. It has been previously reported that S. glomerata bred for QX resistance also have multiple resistance to disseminating neoplasia [10]. This disease is a progressive and highly contagious disorder that is known to cause mass mortalities in farmed bivalves around the world. The causative agent is currently unknown but evidence exists to suggest the disease is caused by a retrovirus during times of environmental stress [11]. The current study was designed to investigate the molecular mechanisms behind the differential resistance to disseminating neoplasia between the two lines of oysters and provides the foundation for genetic research into markers for disease resistance in this economically important bivalve. 2. Materials and methods 2.1. Animals, experimental design and hemolymph withdrawal QX-resistant fourth generation S. glomerata (R) and wild-caught control S. glomerata (W) were obtained from the same farm in Camden Haven, NSW. Three hundred two-year old oysters of equal size from each group were placed within a commercial oyster lease in the Pimpama River, Queensland. Oysters were sampled approximately every 10 days depending on tide and weather. On each

sampling occasion, oysters were washed with seawater to remove accumulated mud (to avoid mud-worm infestation) and mortality was assessed by individually examining each oyster. Eight oysters from each group were sampled at each time point by drilling a hole in the left shell valve and a 0.5 ml hemolymph sample was taken from the adductor muscle using a sterile needle and syringe. Oysters were then tagged (Hallprint Pty Ltd., S Australia) to allow subsequent identification of the fate of sampled individuals, the drill hole was plugged with wax and the oysters returned to the lease. Hemolymph samples were immediately centrifuged in the field and cell pellet resuspended in RNAlater (Sigma) and stored according to the manufacturer’s protocol. Cell free hemolymph samples were immediately frozen on dry ice and stored at 20  C upon arrival at the laboratory. 2.2. Total RNA purification Total RNA was isolated from hemocyte pellets using the RNeasy kit (Qiagen) following the manufacturer’s protocol with on-column DNase digestion using the RNase free DNase set (Qiagen). Quantity of total RNA purified was determined using a spectrophotometer (Nanodrop ND-1000) at 260 nm and quality assessed using the 2100 Bioanalyser and RNA Nano Labchip (Agilent Technologies). RNA was confirmed to be free of DNA contamination by performing a S. glomerata specific 18s rDNA PCR (forward primer 50 -AAAACATCAC TAGGAGAAAC and reverse primer 50 -TCTTACAATCCTTAATAAATAA). 2.3. Identification of immune genes by suppression-subtractive hybridisation (SSH) SSH was performed to generate two libraries of cDNA fragments enriched for genes differentially expressed between QX-resistant S. glomerata (R) and susceptible wild-caught S. glomerata (W). Libraries were generated from three confirmed R and three confirmed susceptible W oysters sampled on day 12 as this was the onset of observed mortality (Results). Oysters were confirmed resistant or susceptible to disseminating neoplasia by the identification tags applied during the non-lethal sampling and subsequently monitoring mortality status of sampled individuals throughout the trial (Section 2.1). Due to the limited starting material, mRNA was reverse transcribed from each oyster and amplified using the Super SMARTÔ cDNA synthesis kit (Clontech) according to the manufacturer’s instructions. Amplified cDNA from each oyster was individually digested according to the manufacturer’s instructions and was pooled in equal proportions (0.66 mg cDNA per oyster) to give a total of 2 mg cDNA for R and W pools, respectively. Forward and reverse subtractions were carried out using the PCR-SelectÔ cDNA subtraction kit (Clontech) following the manufacturers protocol. PCR products generated from SSH were ligated into the pCR4Ò TOPO vector and transformed using TOP10 One ShotÒ chemically competent cells (Invitrogen). Transformed cells were cultured on LB agar plates containing 100 mg L1 ampicillin and 150 clones from each library were randomly picked and cloned inserts amplified by PCR using the universal M13 primer set. Excess primer and nucleotides were removed by enzymatic digestion using exonuclease I and shrimp alkaline phosphatase (Fermentas). PCR products were sequenced uni-directionally using the universal M13F primer by the Australian Genomic Research Facility (AGRF). Sequences were quality scored and visually checked for possible sequencing errors and vector and adaptor sequences were removed using VecScreen (www.ncbi.nlm.nih.gov/VecScreen). Sequences with >90% similarity were grouped into the same contig and regarded as redundant. The sequences were checked for homology to known sequences in GenBank using the BlastX algorithm [12]. ESTs with a significant match (E-values < 1  105, Score > 200) were assigned a putative function using AmiGo (www.geneontology.org) [13]. EST sequences

T.J. Green et al. / Fish & Shellfish Immunology 26 (2009) 799–810

were submitted to the dbEST database (GenBank accession nos. GH612238 to GH612420). 2.4. Quantitative reverse-transcription PCR (qRT-PCR) Nine ESTs, identified from SSH, homologous to genes involved in bivalve immunity (Discussion) were selected for further investigation using qRT-PCR using the gene specific primers listed in Table 1. Primers were designed from EST sequences obtained from SSH using the software package, Primer ExpressÔ v. 2.0 (Applied Biosystems). Total RNA was isolated (Section 2.2) from hemocytes of R and W oysters on days 2, 19, 57 and 95. These four time points were chosen to determine differences in expression over the time course of the experiment. A minimum of four oysters per group at each time point were analysed. First strand synthesis was performed on 100 ng of total RNA using random hexamer primers and SuperScriptÔ III Reverse Transcriptase (Invitrogen) to minimise differences in RT efficiency. cDNA was diluted five-fold prior to amplification and each reaction performed in triplicate. The PCR reaction was performed in a 10 ml reaction using SYBRÒ GREEN PCR Master Mix (Applied Biosystems), 100 nM of each specific primer and 2 ng of cDNA in an ABI 7900HT thermocycler (Applied Biosystems) using an initial denaturation (95  C, 10 min) followed by 45 cycles of a denaturation step (95  C, 15 s) and hybridisationelongation step (60  C, 1 min). A subsequent melting temperature curve of the amplicon was performed. Efficiency of target amplification was optimised prior to running samples for each of the eight primer pairs by trialling four primer concentrations (200, 150, 100 and 50 nM). Constant Ct values were observed at a 100 nM final primer concentration for each of the primer pairs. Amplification efficiency of target and reference genes was calculated using a 5-fold dilution of cDNA and amplification efficiency for each primer pair is listed in Table 1. Expression of target genes was normalised by the house-keeping gene, b-Actin, which was shown to be stable in the current study (Results). Relative expression of the target gene was calculated using the formula: 2(CTtargetCTreference), with the cycle threshold (CT) set at 0.2 for all genes. 2.5. Rapid amplification of cDNA ends and phylogenetic analysis of differentially expressed genes The full-length coding sequences (CDS) were obtained for ESTs homologous to extracellular superoxide dismutase (ecSOD) (EC

801

1.15.1.1) and peroxiredoxin 6 (Prx6) (EC 1.12.1.15) as qRT-PCR results indicated that these two genes were important in disease resistance in S. glomerata (Discussion). Total RNA isolated (Section 2.2) was reversed transcribed and 30 - and 50 -RACE performed following the manufacturer’s protocol (SMART RACE cDNA Amplification Kit, Clontech) using the gene specific primers designed for qRT-PCR (listed in Table 1) and the universal primer supplied in the kit. The generated PCR products were purified and sequenced as above. The CDS was obtained by overlapping sequences determined by 30 - and 50 -RACE. For phylogenetic analysis, amino acid sequences containing full-length open reading frames were selected from GenBank and imported into the software program, Mega v. 4.0 [14]. The number of amino acid sequences used to construct phylogenetic trees were 14 and 16 for ecSOD and Prx6, respectively. Sequences were aligned using the ClustalW algorithm and un-rooted phylogenetic trees were constructed using the neighbour-joining distance method and the outcome of these trees were confirmed with consensus trees constructed using the maximum parsimony method. To derive the confidence value for the phylogeny analysis, bootstrap values are based on 1000 resamplings of the data. 2.6. In-situ hybridisation Hemocyte cell populations comprise of several different cell types [15,16]. Therefore, the differential expression of ecSOD and Prx6 (Results) between R and W S. glomerata could result from different percentages of hemocyte sub-populations that express these two genes between the two groups of oysters as opposed to polymorphic differences within the genes themselves that influence gene expression. Oysters were bled as above (Section 2.1) and hemocytes allowed to adhere to silane coated glass slides for 10 min before being washed in artificial seawater and fixed in 4% paraformaldehyde in 0.1 M PBS for 10 min. Hemocyte cell preparations were permeabilised using 0.3% Triton X-100 in Tris buffered saline (100 mM Tris–HCl, 0.9% NaCl, pH 7.0) for 15 min before hybridisation was carried out overnight at 37  C using 200 ng mL1 of biotinylated oligonucleotide probe (Table 2) in hybridisation buffer containing 5  SSC buffer, 50% formamide, 100 mM DTT and 0.5 mg mL1 heparin. All probes were commercially synthesized and labelled with biotin at the 50 end by the manufacturer (Sigma). Slides were washed twice in 1  SSC buffer for 15 min at 55  C followed by 3 washes in 0.5  SSC buffer for 15 min at 55  C. Slides

Table 1 Primer pairs used and amplification efficiency of primer sets in real-time qRT-PCR expression analysis. Gene

Primer sequence (5’-)

Tm

Amplicon (bp)

Efficiency

Ficolin 4

AACGACAGAAGTGGTGGAAACTG TTGAGTTGTGACAGGCGTTGTAC GCCATTGTTCCACAAAATGTAGATC CAGCTACTTTTTCCGTTGCATAATC TCTTCTCTCGAGGTATTCCGAATG CAGCTGTAAATCCCACCTTTTTG AACTCTACCACGGCGAGCAT CCACGGTCGTCATCATGAAG GAAGGATGGAAGGACGGTGAT CACCTGTGGAAACACCTTCTC AAGAAGAATGCCTGCCACATG GGATGGACAGGATTGAAATTCC GAGAATTCACCAAGTCCTACACACTTC CGCCATCTCTGGACAACGTT AACTGTTTTTACCGGGACGTATG GGTACAGGGCACGTCATTCTC TCGCGATTCTCGGTGTTTC GGACACGGCACTTCATATTGC GTATTGCTGACCGTATGCAGAAAG GGTGGAGCAATGACCTTGATC

59 59 60 59 60 59 58 59 59 59 59 58 59 60 58 58 58 59 59 58

73

2.0

76

2.0

76

2.0

76

2.0

82

1.9

76

2.0

78

2.0

77

2.0

79

1.9

76

2.0

Galactose binding lectin C1q Superoxide Dismutase Peroxiredoxin 6 Metallothionein Small heat shock protein IK cytokine IKB Beta actin

T.J. Green et al. / Fish & Shellfish Immunology 26 (2009) 799–810

Table 2 Oligonucleotide probes used for in-situ hybridisation. Probe

Sequence 5’-

Tm

ecSOD anti-sense ecSOD non-sense Prx6 anti-sense Prx6 non-sense

ATC ACC GTC CTT CCA TCC TTC CTT CCT ACC TTC CTG CCT CTA ATG CTC GCC GTG GTA GAG TT TTG AGA TGG TGC CGC TCG TA

61 61 60 60

were incubated with ImmunoPureÒ avidin, alkaline phosphatase conjugated (Pierce) before slides were developed with 1-stepÔ NBT/BCIP (Pierce). Slides were observed using an Olympus BX41 Microscope and images captured using Olympus DP70 digital camera and annotated using Photoshop (Adobe). To determine the specificity of the ecSOD and Prx6 probes the following controls were conducted: To determine the probes only bind RNA, tissue sections were digested with RNases (40 mg mL1 RNase A, 9.7 mg mL1 Tris–HCl, 372 mg mL1 EDTA) prior to hybridisation with the oligonucleotide probes. To test probe specific binding, hybridisation of the tissue with labelled anti-sense probe (positive probe – reverse compliment) and non-sense probes (negative probe – complimentary and serves as a control for nonspecific binding) were carried out in parallel and in competition with 10  molar concentrations of un-labelled non-sense probe plus the usual concentration of labelled anti-sense probe. 2.7. Native-polyacrylamide gel electrophoresis & staining for anti-oxidant enzyme activity The true biological function of bivalve extracellular superoxide dismutase (ecSOD) protein in the hemolymph is disputed within the literature (Discussion). Attempts to determine the biological function of S. glomerata ecSOD were made. Firstly, the protein concentration of the cell free hemolymph was estimated using the BCAÔ Protein Assay Kit (Pierce) with bovine serum albumin as a standard following the manufacturer’s protocol in the microassay format. Hemolymph protein concentration was determined at each sample point from eight R and W oysters. Electrophoresis was performed in polyacrylamide mini-gels using a Hoeffer SE260 system. Gels, 0.75 mm thick, were prepared using native (no sodium dodecyl sulphate (SDS), no reducing agent) discontinuous Tris-glycine buffered gel with the resolving gels (12% acrylamide/bis) prepared with 1.5 M Tris–HCl, pH 8.8, and the stacking gel (4% acrylamide/bis) prepared with 0.5 M Tris–HCl, pH 6.8. Electrophoresis was carried out at 180 V for 1 h. Aliquots (10 mL) of hemolymph were loaded per lane to determine differences in protein band or enzymatic activity of the hemolymph between R and W oysters. Superoxide dismutase activity was visualised in nativePAGE gels by negative staining using the riboflavin/nitro-blue tetrazolium method [17], with 50 units of superoxide dismutase purified from bovine erythrocytes (Sigma) loaded as a positive control. Hydrogen peroxide generation in gels was visualised by staining with a 1% ferric chloride, 1% potassium ferricyanide solution until blue/green bands appeared on a yellow background [18]. Gels were stained for catalase activity by extensively washing gels in distilled water followed by 0.015% H2O2 in distilled water for 10 min. Catalase activity was visualised as per hydrogen peroxide generation staining with catalase activity appearing as yellow bands on a dark blue/green background [18]. Catalase purified from bovine liver (Sigma) was used as a positive control. Gels were stained for phenoloxidase enzyme activity with 20 mM hydroquinone (1,4benzenediol) and 5 mM 3-methyl-2-benzothiazolinone hydrazone (MBTH) in PBS [8]. Estimation of molecular mass of protein bands was calculated from samples treated with or without dithiothreitol (DDT) and run on SDS-PAGE gels alongside an All Blue Precision Plus

ProteinÔ Standard (BIO-RAD). Quantification of protein band intensity and molecular mass was determined from digital images taken from Coomassie stained gels using the ImageQuant Capture 400 and the software package ImageQuant TL v. 2005 (GE Healthcare). The relative degree of different protein expression between R and W oysters was estimated for each band as the ratio of band intensity of R oysters to W oysters. 2.8. Statistical analysis To determine statistical differences, data was analysed using the computer software package, SPSS v. 11 and differences were considered significant when p  0.05. Two-way analysis of variance (ANOVA) was performed to test for effect of oyster line and day sampled on gene expression for each transcript or hemolymph protein concentration. Tukey’s honest significant difference method for multiple comparisons was used to compare means if significant differences were found. Normality and homogeneity of equal variances were assessed using Levene’s test of equal variances and residual plots. Student’s t-test was used to test for effect of oyster line on protein expression of individual protein bands measure on PAGE gels. Results are presented as the mean  standard error. 3. Results 3.1. Cumulative mortality There was a greater mortality observed within the W oysters (31.7%) over the 118 days of the experiment when compared to R oysters (0.0%) (Fig. 1). The cause of mortality in W S. glomerata was attributed to disseminating neoplasia by examining histology sections. Further details on the onset and prevalence of neoplasia and the occurrence of other infectious agents during this field experiment have been previously reported [10]. 3.2. EST identification and functional annotation A total of 300 clones were processed and 253 sequences (84.3% of the total) were obtained, representing 183 unique genes been identified from 27 contigs and 156 singletons (Table 3). BlastX analysis of ESTs revealed that 110 ESTs matched known genes

100 W

Cumulative Mortality (%)

802

80

R

60

40

20

0 0

20

40

60

80

100

120

Time (Days) Fig. 1. Survival of R and W oyster over the course of the experiment. The cause of mortality in W oyster was due to disseminating neoplasia which is previously reported in Green et al. [10].

T.J. Green et al. / Fish & Shellfish Immunology 26 (2009) 799–810 Table 3 General characteristics of SSH libraries and ESTs obtained from hemocytes of S. glomerata. Forward library; enriched for genes expressed by R hemocytes. Reverse library; enriched for genes expressed by W hemocytes.

Total # clones sequenced Total # sequences analysed Total # of sequences with significant match Contigs Singletons Total # of sequences with no match to database Redundancy (%)

Total

Forward

Reverse

300 253 113 27 156 76 72.3

150 134 65 14 82 35 71.6

150 119 48 13 74 41 73.1

(E value < 105, score > 200) in the GenBank database and of these 64 ESTs could be assigned a putative function. According to the knowledge domains of GOslimmer (www.geneontology.org/) [13], genes involved in catalytic activity, binding, protein binding and metabolic processes constituted the majority of the transcripts in S. glomerata hemocytes. The major difference observed between the two SSH libraries enriched for genes differentially expressed were the number of ESTs homologous to genes involved in peptidase (1.4 and 7.3%) and hydrolase activity (4.2 and 9.4%) for the forward and reverse libraries, respectively. Despite the functional role of many ESTs still being unknown (41.5%), a number of ESTs homologous to genes potentially involved in immunity were identified (Table 4). Table 4 lists the closest match, the database accession number of the matching sequence, the probability and the closest species for each EST. Three ESTs homologous to genes involved in non-self recognition were identified including: Ficolin; an EST with a galactose binding lectin domain; and an EST containing a C1q and tumor necrosis factor domain. Two ESTs involved in detoxifying reactive oxygen intermediates were also identified, an extracellular superoxide dismutase (ecSOD) and peroxiredoxin 6 (Prx6). Other ESTs homologous to genes potentially involved in immunity included interferon induced protein 44, interferon inhibiting cytokine (IK) and inhibitor of the Rel/NFkB signalling pathway (IkB). ESTs identified that were homologous to genes involved in stress and detoxification included a small heat shock protein (sHsP) and metallothionein (Table 4). 3.3. qRT-PCR In order to verify that true differential expression of genes identified by SSH existed between R and W oysters, qRT-PCR was carried out to determine the relative expression of the target genes at four time points over the course of the experiment. The

803

expression of b-Actin was found to be stable (p > 0.05) under the current experimental conditions and so expression of target genes is presented relative to the expression of b-Actin  standard error. Significant differences in expression were observed between R and W oysters for four of the nine transcripts, irrespective of time, indicating that the base-line expression of these four transcripts are constantly up or down-regulated in R oysters. The base-line expressions of ecSOD and sHsP were found to be 3.18- and 2.05-fold higher respectively in R oysters (p < 0.05, Fig. 2A and B). The baseline expressions of Prx6 and IK were found to be 2.75- and 1.5-fold lower respectively in R oysters (p < 0.05, Fig. 2C and D). The expression of ILB was found to significantly change between sampling points (p < 0.05, Fig. 3), but the expression of ILB at each time point was not significantly different between R and W oysters (p > 0.05). No significant differences were found for expression of any of the other transcripts between R and W oysters or between sampling points (p > 0.05) by qRT-PCR. 3.4. Sequence alignment & phylogenetic analysis of ecSOD and Prx6 From SSH and qRT-PCR results, two ESTs homologous to extracellular superoxide dismutase and peroxiredoxin VI were identified as potentially important in immunity of S. glomerata to disease (Discussion). The full-length CDS for ecSOD and Prx6 were obtained using 50 and 30 RACE. The cDNA sequence for S. glomerata ecSOD (GenBank accession no. FJ626709) is 755 bp, containing a 50 untranslated region (UTR) of 46 bp, followed by a 585 bp open reading frame (ORF) which encoded 195 amino acids and a short 30 UTR of 124 bp containing a stop codon (TAA) and a possible polyadenylation signal (ATAAA) 15 bp upstream of the polyadenylation tail (Fig. 4). Analysis of the deduced amino acid sequence with SignalP3.0 (www.cbs.dtu.dk/ services/SignalP/) revealed a putative signal peptide of 31 amino acids suggesting ecSOD is secreted from the cell. The mature peptide consisted of 179 amino acids and has a calculated molecular weight of 19.7 kDa and an isoelectric point of 5.18 (www.angis. org.au/). The mature peptide contains a putative N-glycosylation site (NVS) (www.cbs.dtu.dk/services/NetNGlyc/), suggesting S. glomerata ecSOD is a glycoprotein (Fig. 4). The alignment (using ClustalW) of the selected extracellular Cu/Zn SOD sequences and phylogenetic tree show that S. glomerata ecSOD clusters with other bivalve ecSODs (Fig. 5), with highest homology to Dominin from the Eastern oyster, Crassostrea virginica (79% identity) and Carvortin from the Pacific oyster, Crassostrea gigas (72% identity). Bivalve ecSODs do not cluster with other extracellular SODs from either vertebrates or invertebrates (Fig. 5).

Table 4 ESTs similar to genes directly or indirectly involved in immunity. Forward library; enriched for genes expressed by R hemocytes. Reverse library; enriched for genes expressed by W hemocytes. Best hit

Accession no.

Organism

Library

E-value

Score

Length (bp)

Galactose binding lectin domaina Sialic acid binding lectin Ficolin 4 Superoxide dismutase (Dominin)a Peroxiredoxin 6a Small heat shock protein 24.1 Metallothionein IKB IK cytokine IFI44a GTPase Peptidyl-propyl cis-trans iso. Fibrinogen Fibrinogen-like 2

CAK11506 Q1KM18 Q966W1 BAF30874 CAK22382 Q6KCP0 Q9U1N5 A7UNT3 Q860B9 CAK04349 XP_001493624 ALKXG2 Q19AS1 Q5TYU3

Danio rerio Helix pomatia Halocynthia roretzi Crassostrea virginica Crassostrea gigas Branschiostoma lanceolatum C. gigas Nematostella vectensis D. rerio D. rerio Equus caballus Dermatophagoides farina Branchiostoma belcheri D. rerio

Forward Forward Forward Forward Forward Forward Forward Reverse Reverse Reverse Reverse Reverse Reverse Reverse

4e18 1e5 5e47 2e72 7e122 5e9 1e14 3e37 6e60 3e64 8e40 4e15 7e28 2e37

94 51 196 275 440 63 82 159 233 250 167 83.6 125 148

571 912 667 755 855 292 392 902 735 1671 685 480 407 749

a

Full-length CDS.

A

a

3 2

b 1 0

0.16 0.14

R

B

W

a

0.12 0.10 0.08

b

0.06 0.04 0.02 0.00

R

W

Relative Expression of IK

Relative Expression of sHsP

4

Relative Expression of Prx6

T.J. Green et al. / Fish & Shellfish Immunology 26 (2009) 799–810

Relative Expression of ecSOD

804

0.05 0.04

C

b

0.03 0.02

a

0.01 0.00 0.08

R

W

D

b

0.06 0.04

a

0.02 0.00

R

W

Fig. 2. Differential expression of A) extracellular superoxide dismuatase (ecSOD); B) small heat shock protein (sHsP); C) peroxiredoxin 6 (Prx6); D) interferon inhibiting cytokine (IK) between R and W oysters by qRT-PCR. Expression was presented relative to the expression of the house-keeping gene, b-Actin (mean  SE). Different letters indicate significant differences between oyster groups (p < 0.05, ANOVA).

The cDNA sequence of S. glomerata Prx6 (GenBank accession no. FJ626708) was 855 bp, containing a 50 UTR of 31 bp, followed by an ORF of 686 bp corresponding to 229 amino acids and a 30 UTR comprised of 138 bp containing a stop codon (TAA) and a possible polyadenylation signal (ATAAA) 17 bp upstream of the polyadenylation tail (Fig. 6). The calculated molecular mass of the deduced mature Prx6 was 25.3 kDa and the theoretical isoelectrial point is 7.21 (www.angis.org.au/). No signal peptide was predicted by SignalP3.0. The S. glomerata Prx6 clusters with, and shows high homology to C. gigas Prx6 (94% identity) (Fig. 7).

Coomassie blue stained native-PAGE gels. These four bands comprise 62.6 and 61.7% of the total hemolymph protein content for R and W oysters, respectively. However, R oysters have significantly higher concentration of these four protein bands in their hemolymph compared to W oysters (p < 0.05), which would partly account for the higher hemolymph protein content observed in R oysters. Further characterisation of these protein bands to determine their biological role by staining native-PAGE gels for catalase and phenoloxidase activity proved that these proteins do not have catalase activity and do not correspond to protein bands responsible for phenoloxidase activity. Positive staining for hydrogen

3.5. Hemocyte expression of ecSOD and Prx6

3.6. Protein expression & electophoretic analysis of hemolymph proteins The cell free hemolymph protein concentration was significantly higher in R oysters at each time point during the trial when compared to W oysters (p < 0.01), with the average protein concentration of R and W oysters been 3.1 and 2.6 mg ml1, respectively. The concentration of hemolymph protein of both R and W oysters was also shown to decline significantly over the course of the experiment (p < 0.01). Native PAGE and negative staining for superoxide dismutase (SOD) activity failed to detect true SOD activity in hemolymph samples from R and W oysters (Fig. 9). Interestingly, four pink bands were observed on native gels stained for SOD activity which correspond to the dominant hemolymph protein bands on

0.07 b

0.06

IkB Relative Expression

The differential expression of ecSOD and Prx6 between R and W oysters did not result from differences in hemocyte sub-populations sampled between the two groups of oysters, as in-situ hybridisation performed on adhered hemocyte preparations from R and W oysters revealed 100% of hemocytes sampled were positive for the expression of ecSOD and Prx6 (Fig. 8).

ab

0.05 0.04

ab 0.03

a

0.02 0.01 0.00

2

19

54

95

Day Fig. 3. Differences in the relative expression of inhibitor of Rel/NFkB (ILB) between different sampling points were observed by qRT-PCR (p < 0.05, ANOVA). No difference in the relative expression of ILB was observed between R and W oysters at each sampling point (p > 0.05, ANOVA), but expression was significantly different between day 2 and 95 (p < 0.05, ANOVA). Expression is presented relative to the expression of the house-keeping gene, b-Actin (mean  SE). Different letters indicate significant differences between days (p < 0.05).

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START 1 1 55 4 109 22 163 40 217 58 270 76 325 94 379 113 433 131 487 149 541 167 595 185 649 703 757

aaagcctgtaaccagtgcaccagtctccgtggagctagaaagaagATGAACTGT M N C CTTCTCGTGTTCGTTGTTCTTGTTGGCGCTGCCTTTGCTTCTAAGAAAAACGAA L L V F V V L V G A A F A S K K N E GCCAACATCAACATTTACCTTCACCTCTCTGACGATGCAGCACAGAGTGATGTA A N I N I Y L H L S D D A A Q S D V AATGCAAACTATGCTACCACAATGCACTATGCCCAGTGTGAGATGGAACCCAAC N A N Y A T T M H Y A Q C E M E P N CCCAACCAACCAGCCAGTCTCCATCACCACGTTCACGGAAGCATTGAAATGCCA P N Q P A S L H H H V H G S I E M P CAGCTGGGTGACGGAGAAATGACTATGTCGTTCCACCTGACTGGCTTTAATGTT Q L G D G E M T M S F H L T G F N V AGCGACGACTTCAAGGACCATAACCACGGCCTCCAGATCCACGAGTATGGAGAC S D D F K D H N H G L Q I H E Y G D ATGGAGCACGGCTGTGTCACCATTGGAGAACTCTACCACGGCGAGCATGTCCAG M E H G C V T I G E L Y H G E H V Q GGACACGCTAACCCTGGTGACCTTGGAGATCTTCATGATGACGACCGTGGAAAC G H A N P G D L G D L H D D D R G N GTCACTGATACTAGGAAATTTGATTGGCTCACAATTGGACATGAAGATGGAATT V T D T R K F D W L T I G H E D G I CTTGGACGTTCTCTGGCTATTCTTCAGGGCGATCACACTAGCCACACTGCCATC L G R S L A I L Q G D H T S H T A I ATTGCTTGCTGCGTCATCGGTCGTTCACATGCCCATtaagatgcaatcttattc I A C C V I G R S H A H STOP gtttacaatttgaagacatctcgggggaaacacgtgcaatattcttaaatttac tgacttacttacaaaataaattcataaaaacgttcaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaa

Fig. 4. The cDNA sequence of S. glomerata ecSOD and its deduced amino acid sequence. The start, stop and canonical polyadenylation signal sequence are boxed. The signal peptide predicted using SignalP3.0 is underlined. The predicted glycosylation site is shown in italics.

peroxide revealed that these four bands do produce hydrogen peroxide (Fig. 9). The four protein bands of interest correspond to four bands in SDS-PAGE under non-reducing conditions with apparent molecular mass of 22.1, 22.3, 27.7 and 27.9 kDa (Fig. 9). However, following reduction, these proteins migrate as two thick bands at 31.9 and 36.5 kDa (Fig. 10), suggesting that these protein bands most likely have intramolecular disulfide bonds.

oyster. The current study exploited the difference in survival of two lines of S. glomerata under field conditions during an outbreak of disseminating neoplasia to identify and characterise genes involved in immunity to disease. The multiple-resistance of R S. glomerata to a probable viral disease (disseminating neoplasia) and to an unrelated protozoan disease (M. sydneyi) is noteworthy [10]. 4.1. Identification of genes involved in immunity

4. Discussion Whilst several studies have investigated proteins associated with disease resistance in S. glomerata [5,6,8], to date little is known about the molecular basis of the immune system in this species of

ESTs of S. glomerata homologous to genes known to be involved in immunity in other animals were identified using suppression subtractive hybridisation. SSH is a technique commonly used to identify genes involved in resistance of marine bivalves to disease

Fig. 5. Un-rooted phylogenetic tree based on the representative vertebrate and invertebrate extracellular SOD amino acid sequences. The tree was constructed using the neighbourjoining algorithm in the Mega 4.0 program [14]. Bootstrap values (shown) are based on 1000 resamplings of the data.

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1 1 61 3 121 24 181 44 241 64 301 84 361 104 421 124 481 144 541 164 601 184 661 204 721 781 841

START agactctatcataagtagtagccgttttgaggacgcactgaccactccgtcataATGGTG M V AATTTAGGCGACACTTTTCCGAACTTTGAGGCGGAAACAACTGCTGGAAAAATCAAATTT N L G D T F P N F E A E T T A G K I K F CATGATTTTGTTGGCGACAGTTGGTGCATACTATTCTCCCATCCAGCAGACTACACTCCA H D F V G D S W C I L F S H P A D Y T P GTCTGTACAACAGAACTGGGGAAATGCGTGGAGCTCGAGCCCGAGTTCAAGAAAAGGGGA V C T T E L G K C V E L E P E F K K R G GTCAAAATGATCGCCCTGTCCTGTGATGACGTCCCAAGTCACGAGGGCTGGTCAAAGGAT V K M I A L S C D D V P S H E G W S K D ATTTTAGATTACTGTAAATGTTCCACAGGAAAGCTTCCGTACCCGATCATTTCGGATAAG I L D Y C K C S T G K L P Y P I I S D K AGCCGAGACTTGGCGGTTAAGCTCGGTATGGTAGATCCGGCGGAGAAAGACAATGCTGGA S R D L A V K L G M V D P A E K D N A G CTTCCTCTGACCTGCCGAGCGGTTTTTATAATTGGCCCTGACAAGAAACTGAAATTATCA L P L T C R A V F I I G P D K K L K L S ATGCTGTACCCCGCAACCACAGGACGTAATTTCGCGGAAATTTTGAGGGTAATTGACTCC M L Y P A T T G R N F A E I L R V I D S CTCCAGTTGACCATGAACAAAAAGGTGGCCACCCCTGAAGGATGGAAGGACGGTGATAAG L Q L T M N K K V A T P E G W K D G D K TGTATGGTTTTACCTTCCATCCCCCAGGAAGGTATAGAGAAGGTGTTTCCACAGGGTGTT C M V L P S I P Q E G I E K V F P Q G V ACCGTACAACCCGTACCCTCGGGGAAAGCTTATCTTAGGTTCACACCCCAACCAAAGtaa T V Q P V P S G K A Y L R F T P Q P K STOP ttccatcacaactgcctgtattaaaaatattttgttataaaatagtttcttattttgtac tcgttaggacatgttactaatttactgaataaactattaaactgaatgtgaaaaaaaaaa aaaaaaaaaaaaaaaaaaa

Fig. 6. cDNA sequence of S. glomerata Peroxiredoxin 6 and its deduced amino acid sequence. The start, stop and canonical polyadenylation signal sequence are boxed. The catalytic centres for peroxidase activity (PVCTTE) and phospholipase A2 activity (GDSW) are underlined.

agents or environmental stressors [19–23]. The SSH libraries were constructed using hemocytes as source material for RNA because they are considered to be the main immune cells in oysters [15,16]. Moreover, use of hemocytes lowers the risk of RNA contamination by bacteria and other organisms that reside in the digestive gland. BlastX analysis of sequenced clones from SSH followed by dividing ESTs into functional categories based on the knowledge domains of GOslimmer revealed that the major functional groups were involved in metabolic processes, catalytic activity and protein binding (Supplementary material) which is similar to other studies on marine bivalves [19,24]. The low number of ESTs that could be

assigned a putative function in the current study is a common problem in studies on marine bivalves [24]. 4.2. Differences in the base-line expression of four immune genes The base-line expression of several genes involved in immunity was found to differ significantly between R and W oysters; the base-line expression of ecSOD and sHsP were up-regulated whilst Prx6 and IK were down-regulated in R oysters. Interestingly, qRT-PCR results show that none of the nine transcripts examined between the two lines of oysters embody on/off patterns but are

Fig. 7. Un-rooted phylogeny showing the most likely relationship between representative invertebrate and vertebrate Prx6 amino acid sequences using the neighbour-joining algorithm in the program Mega 4.0 [14]. Bootstrap values (shown) are based on 1000 resamplings of the data.

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Fig. 8. In-situ hybridisation of hemocytes from S. glomerata using biotin labelled oligonucleotide probes. A) Anti-sense ecSOD probe; B) Anti-sense Prx6 probe; C) Non-sense ecSOD probe; D) Non-sense Prx6 probe.

reflected by differences in the levels of transcriptional rate. Differences in the base-line expression of immune genes in other invertebrate lines selected for disease resistance has also been previously observed [20,25]. The partial success of the subtraction efficiency of SSH with only four of the nine ESTs found to be differentially expressed between R and W oysters by qRT-PCR is not uncommon when using the SSH methodology, with efficiency as low as 2% having been reported [26]. The success of the SSH application is limited by factors including complexity of cDNA samples, the number of differences (targets) between cDNA samples [27], the abundance of the

differentially expressed transcript [28] and/or PCR amplification of cDNA due to the limiting material for cDNA subtraction (Section 2.3). In an attempt to maximise the number of differences between R and S cDNA samples, only three R and three W oysters were used for library construction which were confirmed resistant (live at the end of the field study) and susceptible (dead before the end of the field study) to disease, respectively. It may be that there are genuinely very few transcriptional differences between the hemocytes of R and W oysters. This would seem likely as resistance appears to be controlled by several genes, as has been shown previously in the mollusc, Biomphalaria glabrata with respect to the

Fig. 9. Staining of native-PAGE gels for superoxide dismutase (SOD) activity and hydrogen peroxide (H2O2) generation. Panel A) Dominant bands of the cell free hemolymph of R and W oysters stained with Coomassie blue. Panel B) Cell free hemolymph stained for SOD activity. SOD activity is indicated by the clear whitish band on a blue background in control lane. No SOD activity was observed for R and W oysters. However, four pale pink bands were observed on the gel stained for SOD activity, which correspond to the four dominant protein bands from R and W of Coomassie blue stained gels. Panel C) Four dominant protein bands of the cell free hemolymph from R and W oysters produce H2O2 as indicated by the blue/green bands on a yellow background. SOD, SOD purified from bovine erythrocytes; R, resistant S. glomerata; W, wild-caught S. glomerata.

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Fig. 10. SDS-PAGE gel indicating the molecular mass of the major proteins bands of the cell free hemolymph in R and W oysters under non-reducing and reducing conditions. The protein bands that correspond to the pink bands on superoxide dismutase nativePAGE gels are shown by <. M, protein ladder; A, non-reduced R hemolymph; B, nonreduced W hemolymph; C, reduced R hemolymph; D, reduced W hemolymph.

parasitic trematode, Schistosoma mansoni [29–31]. The low success rate may also have been the result of the inability to identify the putative function of a large proportion of the ESTs identified in S. glomerata in the present study. 4.3. Differential expression of two anti-oxidant enzymes potentially involved in respiratory burst The most interesting result from SSH and qRT-PCR was the differential expression of two anti-oxidant enzymes. The base-line expression of an extracellular superoxide dismutase (ecSOD) was higher and the base-line expression of a non-selenium glutathione peroxidase (Prx6) was lower in R oysters compared to W oysters. When the plasma membrane of oyster hemocytes come into contact with pathogens a range of reactive oxygen intermediates (ROIs) that have cytotoxic effects are generated [32,33]. Hypothetically, superoxide anion (O 2 ) is generated through a plasma membrane NADPH oxidase complex [32]. In turn, superoxide is converted to hydrogen peroxide (H2O2) by superoxide dismutase (SODs) and finally peroxidases convert H2O2 into hypochlorite (HOCl) and hydroxyl radicals (OH) [33]. Each oxidant differs in its reactive properties (e.g. ability to cause DNA damage, lipid peroxidation) and the precise role of each particular oxidant in pathogen killing remains unclear. Studies on myeloperoxidase deficient mice indicate the relative cytotoxic roles of specific oxidants are dependent on the species of bacteria or fungi involved [34]. Pathogens also employ a variety of anti-oxidant strategies for survival within their host and effective killing of these pathogens by the host requires the production of the right oxidant at the right time and place [35]. The differential expression of ecSOD and Prx6 in R oysters implies that the conversion of O 2 to H2O2 would occur at a faster rate and to higher concentrations in R oysters due to the increased concentrations of ecSOD, whilst detoxification of the resulting H2O2 would be reduced by the lower concentrations of Prx6 resulting in the accumulation of H2O2. A similar system has been demonstrated to be a major molecular mechanism for resistance of the freshwater snail, B. glabrata to the parasite S. mansoni is due to deficient forms of anti-oxidant enzymes in the respiratory burst pathway [35]. Resistant B. glabrata generate significantly more H2O2 than

susceptible snails [29] and this increased concentration of H2O2 generated by resistant snails has been correlated to higher expression levels of the SOD1 transcript [36]. Expression of the SOD1 transcript was later found to be associated with one allele found in resistant snails [37]. Similarly, the variation in transcript abundance of S. glomerata ecSOD and Prx6 between R and W oysters is likely to be caused by allelic differences rather than differences in the hemocyte sub-populations sampled that express these two transcripts. In-situ hybridisation experiments revealed that all hemocytes sampled from R and W S. glomerata express these two genes. Therefore, one avenue for future studies into DNA markers for disease resistance in S. glomerata could focus on identifying the allelic differences controlling the differential expression of these two genes. The expression of Prx6 in C. gigas was shown to be positively correlated to pollution levels in estuaries in France [38]. Polymorphism analysis of exon 6 of the Prx6 gene in C. gigas revealed 10 different alleles and analysis of these alleles in natural population of C. gigas exposed to different pollution levels revealed that environmental stress selects for heterozygosity of the Prx6 gene [38]. 4.4. Biological function of bivalve ecSOD Identification of unknown ESTs is based on homology to other sequences in the available databases and the biological function of the unknown EST is hypothetically inferred from the known sequence. The hypothesis that one of the mechanisms for resistance in R S. glomerata is their ability to generate hydrogen peroxide at a faster rate and to higher concentrations than W oysters during respiratory burst (Section 4.3) relies on ecSOD being capable of generating hydrogen peroxide, which true superoxide dismutases achieve by conversion of superoxide anion (O 2 ). However, the true biological function of bivalve ecSOD is disputed within the literature [39,40]. Phylogenetic analysis of S. glomerata ecSOD revealed highest homology to an ecSOD characterised from the Pacific oyster, C. gigas (termed CgEcSOD) [39], Cavortin also from C. gigas [40], Pernin from green-lipped mussel, Perna canaliculus [41] and AiEcSOD from the bay scallop, Argopecten irradians [42]. Based on the phylogenetic analysis of the sequences, CgEcSOD and Cavortin are very similar (94% identity) and there is agreement by both authors that they are likely to be the same protein [39,40]. Therefore, in the current manuscript, CgEcSOD and Cavortin are considered to be the same protein and referred to as Cavortin. The high similarity and shared structural characteristics suggest that the ecSOD from S. glomerata and the previously reported proteins share a similar role. However, role and function are disputed: Gonzalez and Colleagues [39] claim that Cavortin is involved in immunity because it contains a putative lipopolysaccharide (LPS) binding site and was reported to display superoxide dismutase (SOD) activity in SDS-PAGE gels. Conversely, Scotti and colleagues [40,41] failed to detect SOD activity in either Cavortin or Pernin on native-PAGE gels stained for SOD activity, and were unable to show that purified Cavortin or Pernin converted O 2 to H2O2. Scotti and colleagues [40,41] propose instead that the biological function of Cavortin and Pernin has evolved to become a metal ion chaperone. The above authors do agree that CgEcSOD, Cavortin and Pernin are the dominant proteins of the hemolymph and the relative mobility in SDS-PAGE gels of each of these proteins decreases under reducing conditions [39–41], suggesting that these proteins most likely have intramolecular disulfide bonds, which was also predicted in the cDNA sequence of AiEcSOD [42]. Translated amino acid sequence analysis of S. glomerata ecSOD revealed a putative signal peptide and this, coupled with the observation that ecSOD is only expressed by hemocytes [39], suggests that ecSOD is secreted from hemocytes into the

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hemolymph. SDS-PAGE showed that the relative mobility of the four dominant protein bands in the hemolymph of S. glomerata also decreased under reducing conditions and the concentration of these four protein bands is significantly higher in R oysters (Fig. 10). Moreover, true superoxide dismutase activity was not detected in the hemolymph of S. glomerata by negative staining in native-PAGE gels (Fig. 9). Interestingly, four pale pink bands appeared on zymograms that correspond to these dominant protein bands of the hemolymph in Coomassie blue stained gels. These protein bands appear to produce hydrogen peroxide as evidenced by the positive reaction of the zymograms in that region (Fig. 9), but not from the dismutation of superoxide anion. Thus, these protein bands may correspond to glycosylated S. glomerata ecSOD. Phylogenetic analysis is supportive of the contention that bivalve ecSOD has evolved to no longer have classical SOD activity as evidenced by the studies on Cavortin and Pernin [40,41]. Further attempts to determine the biological function of these protein bands were made. Previous studies have implicated phenoloxidase as the main immune effector of the hemolymph in S. glomerata [7,8], and that R S. glomerata express significantly higher concentration of the phenoloxidase enzyme in their hemolymph compared to W oysters [7]. Peroxidase and SOD can stain positively for phenoloxidase activity on native-PAGE gels [43]. As no ESTs were identified for phenoloxidase activity in the sequenced SSH libraries, native-PAGE gels were stained for phenoloxidase activity and the four protein bands of interest did not correspond to the protein bands responsible for phenoloxidase activity (phenoloxidase activity is associated with protein bands of molecular mass of c. 130 kDa). Further research is required to determine the biological function of these proteins. As the dominant proteins of the hemolymph they are likely to be involved in respiratory electron transport with hydrogen peroxide generated as a by-product [44]. Recently, the respiratory electron chain in the abalone, Haliotis midae, was shown to be important in immunity with inhibition of the electron transport chain causing a decreased immune response [45]. A central role in immunity for S. glomerata ecSOD is also supported, perhaps through evolution of hydrogen peroxide as by-product of respiratory electron transport. Other studies have also implicated ecSOD as an important gene involved in disease resistance; Cavortin is up-regulated in C. gigas spat selected for increased resistance to Summer Mortality [20], higher expression of Dominin occurs in C. virginica in response to infection with the protozoan Perkinsus marinus [23], and up-regulation of AiEcSOD occurs in A. irradians 12 h post-injection with Vibrio anguillarum [42]. 5. Conclusion This work constitutes the first step towards elucidating the genetic basis of disease resistance in a selected line of S. glomerata and provides a list of ESTs that are homologous to genes thought to be involved in immunity of S. glomerata to disease. Although the putative function and expression of a large proportion of the ESTs identified is still unknown and may play an important role in immunity, four genes were clearly identified to be differentially expressed between R and W oysters. The molecular mechanisms controlling these differences in expression would make a logical starting point for further research into genetic markers for disease resistance in S. glomerata. Acknowledgements The authors would like to acknowledge W O’Connor of NSW DPI, D Turner and T Troop for help in the field and sourcing oysters. We also thank M Dove of NSW DPI and R Hobb for their constructive

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comments. We acknowledge the Australian government for Australian Postgraduate Award to T. Green. Appendix. Supplementary material Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.fsi.2009.03.003. References [1] Berthe FCJ, Roux FL, Adlard RD, Figueras A. Marteiliosis in molluscs: a review. Aquat Living Resour 2004;17:433–48. [2] Nell JA. The history of oyster farming in Australia. Mar Fish Rev 2001; 63:14–25. [3] Nell JA, Perkins B. Evaluation of the progeny of third-generation Sydney rock oyster Saccostrea glomerata (Gould, 1850) breeding lines for resistance to QX disease Marteilia sydneyi and winter mortality Bonamia roughleyi. Aquacult Res 2006;37:693–700. [4] Alvarez MR, Friedl FE, Ruiz Roman F. In vivo chemoactivation of oyster hemocytes induced by bacterial secretion products. J Invertebr Pathol 1995; 66(3):287–92. [5] Bezemer B, Butt D, Nell JA, Adlard RD, Raftos DA. Breeding for QX disease resistance negatively selects one form of the defensive enzyme, phenoloxidase, in Sydney rock oysters. Fish Shellfish Immunol 2006;20:627–36. [6] Peters R, Raftos DA. The role of phenoloxidase suppression in QX disease outbreaks among Sydney rock oysters (Saccostrea glomerata). Aquaculture 2003;223:29–39. [7] Butt D, Raftos DA. Phenoloxidase-associated cellular defence in the Sydney rock oyster, Saccostrea glomerata, provides resistance against QX disease infections. Dev Comp Immunol 2008;32:299–306. [8] Newton K, Peters R, Raftos DA. Phenoloxidase and QX disease resistance in Sydney rock oysters (Saccostrea glomerata). Dev Comp Immunol 2004;28:565–9. [9] Nell JA, Hand RE. Evaluation of the progeny of second-generation Sydney rock oyster Saccostrea glomerata (Gould, 1850) breeding lines for resistance to QX disease Marteilia sydneyi. Aquaculture 2003;228:27–35. [10] Green TJ, Jones BJ, Adlard RD, Barnes AC. Parasites, pathological conditions and mortality in QX-resistant and wild-caught Sydney rock oysters, Saccostrea glomerata. Aquaculture 2008;280:35–8. [11] Barber BJ. Neoplastic diseases of commercially important marine bivalves. Aquat Living Resour 2004;17:449–66. [12] Altchul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acid Res 1997;25:3389–402. [13] Consortium TGO. Gene ontology: tool for the unification of biology. Nat Genet 2000;25:25–9. [14] Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596–9. [15] Canesi L, Gallo G, Gavioli M, Pruzzo C. Bacteria–hemocyte interactions and Phagocytosis in marine bivalves. Microsc Res Tech 2002;57(6):469–76. [16] Pruzzo C, Gallo G, Canesi L. Persistence of vibrios in marine bivalves: the role of interactions with haemolymph components. Environ Microbiol 2005;7(6): 761–72. [17] Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971;44:276–87. [18] Klotz MG, Hutchexon SW. Multiple periplasmic catalases in pathogenic strains of Pseudomonas syringae. Appl Environ Microbiol 1992;58:2468–73. [19] Gestal C, Costa M, Figueras A, Novoa B. Analysis of differentially expressed genes in response to bacterial stimulation in hemocytes of the carpet-shell clam Ruditapes decussatus: identification of new antimicrobial peptides. Gene 2007;406:134–43. [20] Huvet A, Herpin A, De´gremont L, Labreuche Y, Samain J-F, Cunningham C. The identification of genes from the oyster Crassostrea gigas that are differentially expressed in progeny exhibiting opposed susceptibility to summer mortality. Gene 2004;343:211–20. [21] Meistertzheim A-L, Tanguy A, Moraga D, Thebault M-T. Identification of differentially expressed genes of the Pacific oyster Crassostrea gigas exposed to prolonged thermal stress. FEBS 2007;274:6392–402. [22] Pallavicini A, Costa MM, Gestal C, Dreos R, Figueras A, Venier P, et al. High sequence variability of myticin transcripts in hemocytes of immune-stimulated mussels suggests ancient host–pathogen interactions. Dev Comp Immunol 2008;32:213–6. [23] Tanguy A, Guo X, Ford SE. Discovery of genes expressed in response to Perkinsus marinus challenge in Eastern (Crassostrea virginica) and Pacific (C. gigas) oysters. Gene 2004;338:121–31. [24] Tanguy A, Bierne N, Saavedra C, Pina B, Bache`re E, Kube M, et al. Increasing genomic information in bivalves through new EST collections in four species: development of new genetic markers for environmental studies and genome evolution. Gene 2008;408:27–36. [25] Lorgeril Jd, Gueguen Y, Goarant C, Goyard E, Mugnier C, Fievet J, et al. A relationship between antimicrobial peptide gene expression and capacity of a selected shrimp line to survive a Vibrio infection. Mol Immunol 2008; 45:3438–45.

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