Doi:10.1016/j.dci.2003.10.004

Developmental and Comparative Immunology 28 (2004) 565–569 Phenoloxidase and QX disease resistance in Sydney rock oysters Kathryn Newton, Rodney Peters, David Raftos* Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia Received 30 January 2003; revised 19 September 2003; accepted 21 October 2003 QX is a fatal disease in Sydney rock oysters (Saccostrea glomerata) that results from infection by the protistan parasite, Marteilia sydneyi. Since 1997, the New South Wales Fisheries Service has bred S. glomerata for resistance to QX disease. Thecurrent study shows that the QX resistance breeding program has selected oysters with enhanced phenoloxidase (PO) activities.
The third generation of QX-selected oysters was compared to S. glomerata that had never been selected for disease resistance.
PO enzyme assays showed that oysters bred for resistance had significantly higher PO activities than the non-selectedpopulation. There was no difference between populations in the activities of a variety of other enzymes. Native polyacrylamidegel electrophoresis identified a novel form of PO in QX-selected oysters that contributes to their enhanced PO activities. Thisnovel form of PO may represent a specific QX disease resistance factor.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: Saccostrea glomerata; Phenoloxidase; Disease resistance; Oyster oyster production has declined dramatically in recentyears. Approximately 14.5 million dozen S. glomerata The Sydney rock oyster, Saccostrea glomerata were produced annually during the 1970s, but only 7.9 (previously Saccostrea commercialis), is endemic to million dozen were harvested in the 2000/2001 Australia . It has been farmed on Australia’s east coast since the 1870s, and on the west coast since Declining rock oyster production has resulted, at least in part, from mortality associated with infection cornerstone of the Australian oyster industry, rock by two microbial parasites—Mikrocytos roughleyi,which causes Winter Mortality disease, and Marteiliasydneyi, the etiological agent of QX disease .
Abbreviations: DHPPA, 3-(2,4-dihydroxyphenyl)propionic acid; FSW, filtered seawater; MAC, marine anticoagulant; M. sydneyi is a haplosporidean protozoan that invades the digestive gland of susceptible oysters leading to phenoloxidase; PAGE, polyacrylamide gel electrophoresis; QXR3, complete disorganization of the infected tissue .
third generation oysters bred for QX disease resistance.
Mortality associated with QX disease results from * Corresponding author. Tel.: þ61-2-9850-8402; fax: þ61-2- starvation within 60 days of infection. QX disease E-mail address: [email protected] (D. Raftos).
causes up to 98% mortality among rock oysters during 0145-305X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dci.2003.10.004 K. Newton et al. / Developmental and Comparative Immunology 28 (2004) 565–569 late summer (January to April) The only current the Hawkesbury River has never suffered a QX control method is the removal of oysters from rivers disease outbreak. QXR3 oysters were the third prone to QX disease during the infective period.
generation of S. glomerata bred for QX resistance in Recently, we have found that the phenoloxidase the Georges River, NSW. They were kindly supplied (PO) cascade of susceptible oysters is suppressed by Dr John Nell, NSW Fisheries. The parental generation of the QXR strain came from the same invertebrates, the PO cascade represents a critical brood stock as the wild type oysters from the host defense response. Recognition components of the PO cascade can identify a variety of potentially All oysters were collected during the period July – pathogenic microorganisms and activate PO via October, 2002, when there was no active M. sydneyi proteolysis of its zymogen, prophenoloxidase .
infection in the Georges River. Oysters were PO, also known as tyrosinase, is a bifunctional approximately 2-years-old when they were collected.
enzyme with both monophenol monooxygenase After collection, oysters were held for up to 3 (monophenolase) and o-diphenoloxidase (o-dipheno- weeks in 50 l aquaria filled with sea water collected lase) activities Monophenolase activity leads from The Spit, Sydney Harbor. They were maintained to the hydroxylation of substrates such as tyrosine at a constant temperature of 15 8C and were fed to o-diphenols like L-3,4-dihydroxyphenylalanine Aquasonic invertebrate food supplement (1.5 ml/50 l) converts L-DOPA to quinones such as dopaquinone.
This cascade of enzymatic reactions ultimately results in the formation of the pigment, melaninMelanin and other products of the PO Oysters were removed from aquaria 30 min prior to pathway are involved in host defense reactions hemolymph extraction to drain seawater from their such as wound healing, cytotoxicity, phagocytosis mantle cavities. They were then shucked with an oyster knife and the exuding hemolymph was In 1997, the New South Wales (NSW) Fisheries Service began a rock oyster breeding program to For native polyacrylamide gel electrophoresis develop QX disease resistant oysters. This program (native PAGE), 1 ml of hemolymph was removed has been based on interbreeding the survivors of from each oyster and centrifuged for 10 sec at 12,000g annual QX disease outbreaks After just two so that the serum (supernatant) could be collected. To generations of selection, mortality from QX disease prepare hemolymph for microplate enzyme assays, was reduced from more than 90% to 64% Given 2 ml of hemolymph from each oyster was mixed with the apparent association between suppressed PO 2 ml of marine anticoagulant (MAC; 0.1 M glucose, activity and the establishment of QX disease, the 15 mM trisodium citrate, 13 mM citric acid, 10 mM study reported here tests whether oysters bred for QX EDTA, 0.45 M NaCl, pH 7.0) before being centri- disease resistance have enhanced PO activities fuged for 5 min at 3000g. The serum was then removed and placed in separate tubes. Hemocyteswere resuspended in 4 ml of a 1:1 mixture of MACand filtered seawater (FSW; 0.45 mm filter) and allowed to agglutinate. The suspensions were thencentrifuged for another 5 min at 3000g to obtain hemocyte supernatants. Serum and hemocyte super-natants were kept on ice before use. The total protein Two types of S. glomerata, designated QXR3 and contents of sera were measured using a BioRad wild type, were used in this study. Wild type oysters Protein Determination kit with BSA as a standard were collected from commercial oyster leases in Porto (BioRad, Regent’s Park, NSW). Cell numbers in Bay on the Hawkesbury River, NSW. These oysters hemocyte suspensions were calculated with a had not been selected for resistance to QX disease and K. Newton et al. / Developmental and Comparative Immunology 28 (2004) 565–569 acrylamide. Samples consisted of 16 ml of oyster serummixed with 4 ml of 0.35 M Tris-HCl (pH 6.8) containing Two substrates identified by Espin et al. were 10% glycerol. Gels were stained with 20 mM used to test independently for the monophenolase and o- overnight before being rinsed with FSW.
monomethyl ether (Fluka, Switzerland) was used tomeasure monophenolase activity and 3-(2,4-dihydroxy- phenyl)propionic acid (DHPPA; Fluka) was employedto quantify o-diphenolase activity. Both substrates were Enzyme assays of sera or hemocyte supernatants prepared in FSW. The chromogenic nucleophile, from individual oysters were performed in tripli- 3-methyl-2-benzothiazolinone hydrazone (MBTH; cate. Data from triplicates were averaged to provide Sigma Aldrich, NSW) was added to substrate solutions enzyme activities for individual oysters. Mean at a final concentration of 1 mM . In some cases, the values were calculated from the enzyme activities PO-specific inhibitor, tropolone (Sigma Aldrich) was of at least 10 separate oysters ðn $ 10Þ per added (1 mM final concentration) to sera and hemocyte supernatants 15 min prior to the addition of PO Differences between mean enzyme activities were tested for significance using Student’s t-tests. The The activities of six other enzymes were tested frequencies of PO protein banding patterns in using the following substrates (all purchased from different populations were compared by contingency Sigma Aldrich): 4-nitrophenyl acetate (general ester- Chi-squared analysis. Differences were deemed to ase), 4-nitrophenyl N-acetyl-b-D-glucosaminide (N-acetyl-glucosaminidase), 4-nitrophenyl b-D-galac-topyranoside (b-galactosidase), 4-nitrophenyl a-D-maltopentanoside phosphate (alkaline phosphatase) and 4-nitrophenylb-D-triacetylchitotriose (lysozyme). These substrates 3.1. QXR3 oysters have higher phenoloxidase were prepared in FSW and used at a final concen- Enzyme activities were determined spectrophoto- Substantial PO (monophenolase and o-dipheno- metrically in 96 well microtest plates (Sarstedt, lase) activity was detected by microplate assays of Technology Park, SA). In each microplate well, serum and hemocyte supernatants from QXR3 and 100 ml of serum or hemocyte supernatants were wild type oysters. Three to four times more PO mixed with 100 ml of substrate solution. Absorbance activity was evident in hemocytes when compared to at 490 nm (substrates containing MBTH) or 415 nm serum ðp , 0:001Þ: The PO-specific inhibitor, tropo- (4-nitrophenol based substrates) was measured using a lone, eliminated activity against the monophenolase microplate spectrophotometer (BioRad). Control wells susbtrate, hydroquinone, and the o-diphenolase containing 100 ml of substrate solution mixed with substrate (DHPPA) (p . 0:05 vs substrate only 100 ml of MAC:FSW were included in all plates. Data controls). This confirms that the conversion of both were corrected for spontaneous hydrolysis of substrates measured true PO activity.
Microplate assays also revealed a significant difference in PO activity between the two oyster populations. Sera and hemocyte supernatants fromQXR3 oysters had approximately two times more Differences between the PO proteins of QXR3 and monophenolase and o-diphenolase activity than the wild type oysters were identified by native PAGE according to the method of Nellaiappan and Vinayakam Unlike PO, the activities of six other enzymes Native PAGE gels had a lower resolving layer of (esterase, N-acetyl-glucosaminidase, b-galactosidase, 5% acrylamide and an upper stacking layer of 4% a-amylase, alkaline phosphatase and lysozyme) in K. Newton et al. / Developmental and Comparative Immunology 28 (2004) 565–569 hemocyte supernatants did not differ significantlybetween QXR3 and wild type oysters ðp . 0:05Þ:There was also no significant difference between thetwo oyster populations in the total protein content ofserum or in the number of cells in hemocytesuspensions ðp . 0:05Þ: Serum from QXR3 oysterscontained 38 ^ 3 mg protein/ml, and their hemocytesuspensions averaged 5.4 ^ 0.9 £ 105 cells/ml. Wildtype oysters yielded serum samples with total proteincontents of 36 ^ 4 mg protein/ml, and their hemocytesuspensions contained 5.3 ^ 0.7 £ 105 cells/ml.
Fig. 2. Monophenoloxidase activities in hemocyte supernatants 3.2. QXR3 oysters express a novel form of PO from individual QXR3 oysters (identified by arbitrarily assignednumbers) that had been shown by native PAGE to express either one Native PAGE identified two distinct PO protein or two forms of PO. Activities are shown as change in absorbance at Twenty six percent (12/46) of QXR3 oysters exhibitedtwo distinct PO bands after native PAGE. Only the percent (1/38) of wild type oysters exhibited two PO lower of these two bands could be detected among the bands, whilst the remainder (97%) had only the lower PO band. Chi-squared analysis confirmed that there was a significant difference in the frequency of thetwo PO banding patterns between QXR3 and wild typeoysters ðp , 0:001Þ: identified six oysters that exhibited two PO proteinbands after native PAGE The mean POactivity in hemocyte supernatants from these sixoysters (DOD higher ðp , 0:001Þ than that for the remaining 14oysters (DOD one PO protein (lower band) after native PAGE.
This study has shown that oysters selected for resistance to QX disease have significantly higher PO(monophenolase and o-diphenolase) activities thanwild populations cannot be explained by variation in totalprotein expression or circulating hemocyte frequencies.
Wild type and QXR3 oysters had very similar total Fig. 1. A. Phenoloxidase staining patterns of serum from QXR3 that protein contents in their serum and comparable numbers had been subjected to native PAGE and stained with hydroxyqui- of cells in their hemocyte suspensions.
none and MBTH. Two phenotypes, one PO band (lane 1) or two PO We have also demonstrated that increased PO bands (lane 2) were evident among QXR3 oysters. B. Percentage of activity in QXR3 oysters is associated with the QXR3 and wild type oysters in which two PO bands could be expression a novel form of PO protein. Native PAGE detected by native PAGE (n ¼ sample size, bars ¼ binomialstandard error).
showed that 26% of QXR3 oysters have a second form K. Newton et al. / Developmental and Comparative Immunology 28 (2004) 565–569 of PO, in addition to the enzyme that is common [2] Nell J. Oyster industry report. In: Status of fisheries resources among wild type oysters. Only 3% of wild type oysters 2001/2002, Sydney: NSW Fisheries; 2002. p. 103.
exhibited this additional form of PO. Expression of [3] Perkins FO, Wolf PH. Fine structure of Marteilia sydneyi— haplosporidan pathogen of Australian oysters. J Parasitol the second PO protein is clearly associated with the increased enzyme activities of QXR3 oysters. The [4] Kleeman SN, Adlard RD, Lester RJG. Detection of initial mean PO activity of QXR3 oysters bearing the addition infective stages of the protozoan parasite Marteilia sydneyi in PO protein was almost three times greater than that Saccostrea glomerata and their development through to of oysters exhibiting a single (wild type) form of sporogenesis. Int J Parasitol 2002;32:767 – 84.
[5] Peters R, Raftos D. The role of phenoloxidase suppression in QX-disease outbreaks among Sydney rock oysters (Saccostrea All of this evidence suggests that the QX disease glomerata). Aquaculture 2003;223:29– 39.
resistance breeding program has selected oysters [6] Asokan R, Arumugam M, Mullainadhan P. Activation of bearing an additional PO protein that enhances their prophenoloxidase in the plasma and haemocytes of the marine PO enzymatic activities. That selection does not mussel Perna viridis Linnaeus. Dev Comp Immunol 1997;21: appear to be a chance effect of inbreeding that might have occurred during the intensive develop- [7] So¨derhall K. The prophenoloxidase activating system. In: So¨derhall KS, Iwanga S, Vasta G, editors. New directions in ment of the QXR strain. The activities of other invertebrate immunology. Fair Haven: SOS Publications; enzymes (esterases, b-galactosidase, a-amylase, N-acetyl-glucosamidase, alkaline phosphatase and [8] So¨derhall K, Cerenius L. Role of the prophenoloxidase lysozyme) do not differ significantly between the activating system in invertebrate immunity. Curr Opin QXR and wild type populations, indicating that the breeding of QXR oysters has not resulted in chance [9] Chase MR, Raina K, Bruno J, Sugumaran M. Purification, genetic drift. It is also unlikely that the appearance characterisation and molecular cloning of prophenoloxidasefrom Sarcophaga bullata. Insect Biochem Mol Biol 2000;30: of the novel PO protein in QXR3 oysters simply reflects the genetic background of that population.
[10] Dicko M. Zymography of monophenolase and o-diphenolase The QXR3 and wild type populations are derived activities of polyphenol oxidase. Anal Biochem 2002;306: from the same original brood stock and so have the same genetic origin. The lack of inbreeding effects [11] Prota G. Melanins and melanogenesis. San Diego: Academic and the common genetic origin of the QXR and [12] Ashida M, Brey P. Role of the integument in insect defence: wild type populations implies that the increased PO prophenoloxidase cascade in the cuticular matrix. Proc Natl activity of QXR3 oysters represents a specific, directionally selected trait that protects Sydney [13] Gillespie JP, Kanost MR, Trenczek T. Biological mediators of insect immunity. Ann Rev Entomol 1997;42:611– 43.
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for their generosity in the provision of oysters used [17] Espin JC, Garcia-Ruiz PA, Tudela J, Varon R, Garcia- Canovas F. Monophenolase and diphenolase reaction mech- anisms of apple and pear polyphenol oxidases. J Agric FoodChem 1998;46:2968– 75.
[18] Kahn V, Andrawis A. Inhibition of mushroom tyrosinase EC- 1.10.3.1 by tropolone. Phytochemistry 1985;24:905 – 8.
[19] Nellaiappan K, Vinayakam A. A method for demonstrating [1] Nell J. Farming the Sydney rock oyster (Saccostrea commer- prophenoloxidase after electrophoresis. Biotech Histochem cialis) in Australia. Rev Fish Sci 1993;1:97 – 120.

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