DISEASES OF AQUATIC ORGANISMS
Vol. 40: 137-146.2000 ~ Published March 14 Dis Aquat Org
Molecular detection of Marteilia sydneyi, pathogen of Sydney rock oysters
Sarah N. ~leeman'l*,Robert D. ~dlard~
'~epartmentof Parasitology, University of Queensland, Brisbane, Queensland 4072, Australia '~rotozoa Section, Queensland Museum, PO Box 3300, South Brisbane, Queensland 4101, Australia
ABSTRACT: The life cycle of Marteilia sydneyi, the aetiological agent of QX disease in the Sydney rock oyster Saccostrea commercialis, is not known. We have developed and optirnised 2 diagnostic assays, the polymerase chain reaction (PCR) and in situ hybridisation, for use in investigating the role of pos- sible alternative hosts in the life cycle of this pathogen. PCR primers, designed within the ITS1 rDNA of M. sydneyi, amplified a 195 bp fragment. Sensitivity of the PCR assay was assessed using DNA extracted from known numbers of sporonts purified from infected oyster digestive gland. DNA equiva- lent to 0.01 sporonts was detectable following agarose gel electrophoresis. The potential inhibitory effect of the presence of host DNA on the PCR assay was tested by the addition of oyster genornic DNA during amplification. Concentrations of host DNA in excess of 50 ng per 20 p1 reaction reduced the sensitivity of the test. Environmental validation of the PCR assay was demonstrated by the amplifica- tion of M. sydneyl DNA from 50 ng of genomic DNA extracted from QX-infected oysters. A DNA probe was constructed using the M. sydneyi unique primers and was able to detect 10 pg of M. sydneyi PCR amplified DNA in dot-blot hybridisations. The probe hybridised with presporulating and sporulating M. sydneyi stages in paraffin sections of oyster digestive gland. No non-specific binding was observed. Hybridisation consistency and signal intensity decreased as sporonts matured. While the high sensitiv- ity and specificity of the PCR test will allow rapid screening of large numbers of potential alternative hosts for the presence of parasite DNA, it does not actually identify ~nfectivestages. In situ hybrid- sati ion conducted on paraffin sections will determine the location of the parasite within the host for n~orphologicalcharacterisation.
KEY WORDS: Marteilia sydneyi . PCR primers . DNA probe . In sjtu hybridisation . Saccostrea com- mercialis . rDNA gene sequence
INTRODUCTION limits and prevent further geographic spread of the disease, potential control is hindered by the incom- Marteilia sydneyi,the aetiological agent of QX dis- plete understanding of the life cycle of this pathogen. ease, is recognised as the most pathogenic parasite of Development and ultrastructure of presporulating and the Sydney rock oyster Saccostrea commercialis in sporulating stages in the oyster host are described culture on the east coast of Australia, where stock mor- (Perkins & Wolf 1976); however, the stage infective to talities in excess of 90% have been recorded. Prior to the oyster and its origin are unknown. Failure to infect 1994, QX infections were recorded from the Great oysters experimentally with mature spores (Lester 1986) Sandy Strait (25'30's) south to the Macleay River and recent evidence which suggests a limited period (31" S),after which time its range had extended further of viability of spores in seawater (Wesche et al. 1999) south to include the Georges River estuary (34"s) have implicated the involvement of an intermediate (Adlard & Ernst 1995). Although current management host. strategies exist to keep stock losses within manageable Traditionally, detection of QX in oyster tissue in- volved microscopical examination of histological sec- tions. Wet smears of hepatopancreas, in which refrac- tile bodies in sporonts are characteristic of infection,
O Inter-Research 2000 Resale of full article not permitted 138 Dis Aquat Org 40: 137-146,2000
provided a simple and rapid diagnosis when sporonts for 6 to 24 h in 10% buffered formalin, dehydrated in were present. Current diagnosis is based on the exam- alcohol and embedded in paraffin. Histological sec- ination of Hemacolor (Merck)-stained imprints of he- tions (6 pm) were stained in haematoxylin and eosin patopancreds which enable rapid detection of all de- (H&E) to confirm presence or absence of M. sydneyi velopmental stages, including presporulating stages, in adjacent sections. undetectable in wet preps, within a few days of initial DNA extraction and purification. Genomic DNA was infection. These techniques cannot identify unambigu- extracted from purified sporonts, Marteilia sydneyi in- ously stages of Marteilia sydneyi of unknown morpho- fected Saccostrea commercialis digestive gland and logy that may occur in alternate hosts, highlighting the sperm of uninfected oysters by digestion in a solution need for an appropriate diagnostic tool. An indirect of extraction buffer (100 mM Tris, pH 8.0, 100 mM fluorescent antibody test (IFAT) was developed (Roubal EDTA, pH 8.0, 100 mM NaCl), 10% SDS and Pro- & Lester 1987) but was considered an unreliable guide teinase K (20 mg ml-') at 56°C for 4 to 24 h. Remaining to infection as it did not react to the contents of the proteins and polysaccharides were removed by phenol/ spore (Roubal et al. 1989), the most likely stage to be chloroform/isoamyl alcohol extraction and nucleic present in an alternate host. acids recovered by ethanol precipitation (see Sam- DNA techniques display levels of sensitivity and brook et al. 1989). DNA concentration and purity were specificity, irrespective of life cycle stage, required for estimated by measuring the 260/280 optical density such a test. PCR and in situ hybridisation assays have ratio of a solution following RNase treatment (0.2 pg proven to be usefu! toc!s fer diszasc detection in RNase per pi oi yenomic DNA). aquatic animals (Adlard & Lester 1995, Chang et al. Primer synthesis. A reverse primer, based on the 1996, Durand et al. 1996, Lo et al. 1997, Wang et al. putative first internal transcribed spacer (ITS1) se- 1997) and are considered suitable tools for life cycle quence given by Anderson et al. (1995), was designed studies (Fong et al. 1993, Stokes & Burreson 1995, and designated PR02. It was synthesised from the re- Stokes et al. 1995). Polymerase chain reaction (PCR) verse complement of PRO1 (Anderson et al. 1995) such primers developed by Anderson et al. (1995) were that PR02 = 5'-TCA AGG GAC ATC CAA CGG TC-3'. found to be specific to Marteilia sydneyi. However, This primer was then used in a PCR amplification with their location in the rDNA gene cluster was not con- a forward primer designated RA2 (5'-GTC CCT GCC firmed and subsequent analysis revealed low levels of CTT TGT ACA CA-3') with annealing site in a con- sensitivity in PCR (Adlard unpubl. data). served region of the small subunit (SSU), 180 nucleo- In this paper we describe the development and opti- tide bases upstream of the boundary between the SSU misation of 2 diagnostic assays based on PCR and in and ITS 1. situ hybridisation, utilising PCR primers that amplify This PCR produced an amplified fragment of 430 bp specific DNA regions within the ribosomal DNA clus- when compared on an agarose gel with a molecular ter of Marteilia sydneyi, which can then be applied to weight standard (100 bp ladder, GibcoBRL). The nu- investigate the role of possible alternate hosts in the cleotide sequence was determined for this fragment life cycle of this important pathogen. using the same primers as sequencing primers, then compared for homology with known SSU sequences (Cryptocaryon irntans, and compared using Blast rou- METHODS tine on GenBank) to confirm its location in the rDNA cluster. To increase the species specificity for hybridis- Specimen preparation. Oysters were tested for the ation of a PCR-generated probe, a second forward presence of Marteilia sydneyi by microscopical exami- primer designated LEG 1,5'-CGA TCT GTG TAG TCG nation of Hemacolor (Merck)-stained imprints of oyster GAT TCC GA-3', was designed from this sequence digestive gland. Infected oysters were classified as within the original fragment, but positioned 48 nucleo- having: (1) presporulation stage of infection if plas- tide bases downstream of the SSU/ITS1 boundary. moaia and secondary cells were identified i.n imprints; PCR.PCR was performed under the following reac- and (2) progressi.ve stage of infection if all develop- tion parameters unless otherwise stated, expressed as mental stages (plasmodia, secondary cells, immature final concentrations: MgC12 2 mM; buffer 67 mM Tris- and mature sporonts) were present. Sporonts of M. HCl, 16.6 mM (NH,)SO,, 0.45% (v/v) Triton X-100; sydneyi were purified from homogenised digestive dNTP's 200 mM; primers 10 pm01 each; Taq polymer- gland of infected oysters by centrifugation on discon- ase (Boehringer, Mannheim) 3 U; DNA 50 ng, ultra- tinuous sucrose and Percoll gradients as described pure water to 50 p1 total. Thermal cycling parameters by Mialhe et al. (1985). Tissue samples of uninfected were as follows: denaturation at 95OC for 60 s; primer oysters, oysters with presporulation stages of QX and annealing at 55'C for 30 S; chain extension at 72°C for oysters with progressive stages of infection were fixed 60 S, repeated for 30 cycles with a final cycle incorpo- Kleeman & Adlard: Molecular detect~onof Marteiliasydneyi 139
rating a 7 rnin extension. Electrophoresis of amplified Mannheim) in a spot test on nylon membranes accord- products was conducted through submarine agarose ing to the methodology in the application manual. gels (1.2% [w/v] agarose, 1.2 p1 ethidium bromide DIG-labelled DNA probe sensitivity. Sensitivity of [l0 mg ml-' w/v] in 20 m1 total, for 30 rnin at 100 V per the probe was determined in dot-blot hybridisations. 20 mA) and examined and photographed under ultra- Serial dilutions from 10 ng to 1 pg of Marteilia sydneyi violet light. A molecular weight standard (100 bp PCR cloned DNA were heat-denatured and spotted ladder, Gibco BRL) was used to estimate the size of onto nylon membranes then fixed by UV crosslinking products. for 3 min. Membranes were prehybridised in 3 X SSC DNA sequencing. Amplified products were purified (20 X SSC = 3 M NaCl; 0.3 M Na-citrate, pH 7.0), using Qiaquick PCR purification spin columns (Qiagen 50% formamide, lx Denhardt's solution and 0.5 mg ml-' Inc). The concentration of the purified product was heat denatured herring sperm DNA at 37°C for 2 h. assessed by comparison on an agarose gel with DNA of The prehybridisation buffer solution was replaced with a known mass (Mass ladder, GibcoBRL), then an esti- hybridisation buffer (3 X SSC, 50% formamide, lx mated 50 ng of purified PCR product used in a cycle Denhardt's solution and 0.5 mg ml-' heat denatured sequencing reaction employing dye-nucleotide ter- herring sperm DNA and 5 % Dextran sulfate) contain- minators (ABI, Prism Ready Reaction Terminator Kit). ing 50 ng ml-' DIG-labelled DNA probe and incubated Sequencing reaction products were purified with phe- overnight at 42°C. Removal of unhybridised probe was nol/chloroform and sequenced on an AB1 373A auto- achieved by two 5 rnin washes in 2 X SSC at room mated sequencer. Sequences were manipulated and temperature and two 15 rnin washes at 42°C with 0.1 X analysed using the computer software ESEE (Cabot & SSC. Following equilibration in maleic acid buffer Beckenbach 1989). (100 mM maleic ac~d,150 mM NaC1, pH 7.5), mem- PCR sensitivity. Purified Plarteiha sydneyi sporonts branes were blocked for 30 rnin at room temperature in were counted using a Nebauer haemocytometer, di- blocking buffer (maleic acid buffer plus 1 % blocking luted to a working concentration of 1 X 106 sporonts reagent; Boehringer, Mannheim). Membranes were and DNA extracted following the previously described incubated at room temperature for 30 rnin with anti- method. Serial dilutions from 100 to 0.001 sporont dioxygenin-alkaline phosphatase antibody (Boehringer, DNA equivalents (63.8 pg to 0.638 fg) were used as Mannheim) diluted 1:5000 in blocking buffer followed template in 20 p1 PCR reactions. To determine if the by removal of unbound antibody with two 15 min sensitivity of the PCR reaction was compromised by washes in washing buffer (maleic acid buffer + 0.3% the presence of host tissue, 50, 100, 200 and 400 ng of Tween 20). After equilibration in detection buffer genomic DNA extracted from uninfected oyster sperm (100 mM Tris-HC1, 100 mM NaCI, 50 mM MgC12, was added to 20 p1 PCR reactions containing serial pH 9.5) the membrane was incubated at room temper- dilutions of 100 to 0.01 sporont DNA equivalents. Envi- ature in the dark for 4 to 5 h in 5-bromo-4-chloro-3- ronmental validation of the PCR reaction was carried indolyl phosphate/nitro blue tetrazolium (NBT/BCIP) out on 50 ng of genomic DNA extracted from wild diluted in detection buffer. The reaction was stopped oysters assessed to have: no parasite presence; low with a TE buffer wash. (
cooled on ice for 5 min and allowed to hybridise over- M.sydney1 GAGGGTGGAAAAGTCGTTACACGGTCTCCGTAGGTGAACCTGCG night in a humid chamber at 42OC. Post hybridisation C.irritans ....AA... G ...... A ... A. .. T......
washes including 2 X SSC at room temperature, twice for t 3'189 1 5'ITSls 5 min, and 0.1 X SSC at 42"C, once tor 10 min were car- M.sydneyi GGGGGATCACCCAATGAGATATACCGTCGCCCCATGTGCGATGG C.lrritans .AA...... ried out, followed by equilibration in maleic acid buffer. DIG-labelled probe detection included blocking sections LEGl with 500 p1 blocklng buffer at 37OC for 15 min followed M.sydney3 CGACATCTTCGCGATCGATCTGTGTAGTCGGATTCCGATTTGGT by incubation for 1 h at 37°C with 500 p1 of dilute anti- dioxygenin-alkaline phosphatase conjugate (1:1000 in M.sydneyi CCTCGTCGTCGAAATACGGATCTTCGACCATAGCGGTCGCGTCG blocking buffer). Unbound antibody was removed with two 5 rnin washes in washing buffer and one 5 min wash M.sydneyi ATGTTCGTACTTCGGAGGATCGGTCACTCATGTTCGTCATCCGT in detection buffer. NBT/BCIP was diluted in detection buffer plus 1 % polyvinyl alcohol and 1 mM levamisole M.sydneyi GACGTCTTTCCGATCGGTCCTTTCCATGGGACGCCATCCTATCG and 100 p1 of the colour solution added to the tissue and PR02 incubated at room temperature in the dark for 4 to 5 h. M.sydneyi TATAGTCGATGTACGACCGTTGGATGTCCCTTGTCCCTTGA The reaction was stopped with a TE buffer wash. Slides were washed in ddH20 and stained for 1 rnin in Bismark Fig. 1. Ribosomal DNA nucleotide sequence of the ITSl and flanking 18s region of Marteiliasydneyiisolated from Saccos- brown Y followed by ethanol dehydration and mounted trea commerciaLis (GenBank accession number: Ranklt275614 in DPX via to!uene. Hybridisation conditions were AF159248). The sequence of the PCR primers LEGl and optimised by varying the concentrations of Proteinase K PR02 are underlined. The 195 bp DNA probe sequence is and the DIG-labelled DNA probe. Negative controls marked in bold. Alignment with Cryptocaryon irritans data included samples without the digoxigenin-labelled for 18s taken from Diggles & Adlard (1995). Periods indicate homologous base probe in the hybridisation mixture.
DNA probe sensitivity RESULTS Fifty ng ml-l of the DNA probe detected consistently Extended sequence data of the putative ITSl se- 10 pg of PCR amplified Marteilia sydneyi DNA in dot quence given by Anderson et al. (1995) were aligned blot hybridisations (Fig. 5). with known sequences (Cryptocaryon irritans, see Diggles & Adlard 1995), and confirmed their position in the rDNA gene cluster (Fig. 1). The forward primer In situhybridisation LEGl and the reverse primer PR02 provided the basis for a specific PCR amplification of a 195 bp fragment The DNA probe hybridised with presporulating and within the ITSl sequence of Marteilia sydneyi (Fig. 1). sporulating stages of Marteilia sydneyi in paraffin-em-
PCR sensitivity
The PCR assay was found to be sensitive enough to repeatedly amplify a DNA concentration equivalent to 0.01 sporonts (6.38 fg) (Fig. 2).Ad.dition of 50 ng of host DNA to the reaction did not compromise the sensitivity of the test; however, the intensity of the signal was reduced (Fig. 3A). Further reductions in signal inten- siiy ana inhibition of the PCR were found at host DNA concentrations greater than and equal to 100 ng. In the presence of 100 and 200 ng of host DNA the PCR test Fig 2 Agarose gel electrophores~sassessment of the sens~t~v- ~tyof the pnmer palr LEGl/PR02 In PCR on vanous concen- was able to detect 1 and 100 sporont DNA equivalents, tratlons of A/lartell~a sydneyl DNA extracted from sporonts respectively (Fig. 3B,C). One hundred sporont DNA punfled from Infected Saccostrea cornrnerclalls Lane 1, equivalents were not detectable in PCR after addition 100 bp ladder, lane 2, 100 sporont DNA equlvalents (63 8 pg), of 400 ng of host DNA (Fig. 3D). Marteilia sydneyi lane 3 10 sporont DNA equlvalents (6 38 pg), lane 4, 1 spo- ront DNA equivalent (0 638 pg), lane 5, 0 1 sporont DNA infections in Saccostrea commercialis, ranging in in- equivdlents (63 8 fg), lane 6, 0 01 sporont DNA equlvalents tensity from low to high, were detectable in PCR. Unin- (6 38 fg), lane 7, 0 001 sporont DNA equivalents (0 638 fg), fected oysters yielded no result (Fig. 4). lane 8, no DNA control Kleeman & Adlard: Molecular detection of Marteiha sydneyi 141
Fig 4. Environmental validation of the PCR test on QX- infected Saccostrea commercialiswith various intensities of infection. Lane 1. 100 bp ladder; lane 2, high infection inten- sity; lane 3, medium infection intensity; lane 4, low infection intensity; lane 5, uninfected oyster; lane 6, no DNA control
from an individual with a presporulation stage of in- fection were easily located following probe detection (Fig. K), but were difficult to locate in histology at low power (200x) (Fig. 6D).Negative control tissue sam- ples known to be unparasitized showed no non-specific hybridisation to oyster tissue (Fig. 6E). No signal was detected in M. sydneyi-infected tissue where the di- oxygenin-labelled probe was omitted (Fig. 6F). Hybridisation consistency and signal intensity varied with different developmental stages of the parasite. Plasmodia and secondary cells showed strong positive results in repeated assays (Fig. ?A).Immature sporonts exhibited consistent strong hybridisation signals lo- calised within the spores (Fig. ?B). As the sporont matured the signal decreased progressively and hy- bridisation was detected inconsistently in very mature stages. Hybridisation was concentrated in the inner- most and intermediate sporoplasms in mature spores with some reactivity in the spore wall (Fig. ?C).No sig- Fig. 3. Assessment of the sensitivity of the primer pair nal was observed in the refringent granules (Fig. ?B,C). LEGl/PR02 in. PCR on various concentrations of Marteilia Mature spores in sporonts shed into the lumen of the sydneyi DN.4 extracted from purified sporonts in the pres- ence of increasing concentrations of genomic DNA extracted digestive tubules hybridised strongly and consistently from uninfected Saccostrea commercialis.Ethidium bromide (Fig. 8A). The corresponding region in adjacent sec- staining of M. sydney~an~plification products in the presence tlons in histology confirmed maturity of shed spores of (A)50 ng; (B)100 ng; (C)200 ng; (D) 400 ng of S. commer- cialis genomic DNA. Lane 1, 100 bp ladder; lane 2, 100 spo- ront DNA equivalents (63.8 pg); lane 3, 10 sporont DNA equi- valents (6.38 pg); lane 4, 1 sporont DNA equivalent (0.638 pg); lane 5, 0.1 sporont DNA equivalents (63.8 fg);lane 6, 0.01 spo- ront DNA equivalents (6.38 fg) bedded tissue prepared from an oyster determined to have a progressive stage of infection, with negligible background hybridisation (Fig. 6A). Parasite identity Fig. 5. Sensitivity of the DIG-labelled DNA probe in dot blot and location were confirmed in the adjacent section hybridisation. From left to right each dot contains: (1) 10 ng; after staining with H&E (Fig. 6B). Individual plasmodia (2)1 ng; (3)100 pg; (4)10 pg; (5) 1 pg of PCR-amplified and secondary cell stages in oyster tissue prepared Marteilia sydneyi SSU rDNA
Kleeman & Adlard: Molecular detection of Marteilia sydneyi 143
Fig. 6. Detect~onof Marteilia sydneyi DNA in tissue sectlons of the digestwe gland of infected Saccostrea commercialis by in situ hybridisation with adjacent sections stained in H&E (A-D). Negative controls for in situ hybridisation (E-F). (A) In situ hybrid- isation with plasmodia (arrow) in the intestinal epithelia and spores within sporonts (1 of many sporont clusters indicated by the arrowhead) in the digestive tubules of a heavily infected oyster. (B) H&E-stained section of the same individual as in (A).The same regions are indicated by arrows. (C) In situ hybr~disationwith presporulating stages (arrows) in the digestive tubules of a lightly infected oyster. (D)H&E-stained section of the same individual as in (C).Scale bar A-D = 100 pm. (E)Absence of reaction with in situ hybridisation using the DNA probe with uninfected oyster tissue. Scale bar = 120 pm; (F) In situ hybridisation with the probe omitted with tissue from the same oyster as in (A) & (B).Some of the many sporonts within sporangiosori are indicated by arrows. Scale bar = 40 pm. Dt: digestive tubule; Ie: intestinal epithelia
(Fig. 8B). Modification of the probe concentration for pathogen among related species. Results of the present optimisation of the hybridisation procedure did not im- study indicate that the first 2 of these requirements at prove the likelihood of hybridisation in mature spores least have been met. The third remains untested, al- and concentrations of Proteinase K in excess of 10 pg though it is implied through the relatively high muta- ml-Lcompromised tissue morphology. tion rates in ITS regions (Li & Graur 1991), which in turn may confer inherent specificity to the assay. The primer pair LEGl/PR02 exhibited high detec- DISCUSSION tion sensitivity in PCR able to detect quantities of ge- nomic Marteilia sydneyi DNA equivalent to 0.01 spo- Assays developed for diagnosis of cryptic parasite ronts (6.38 fg) in the absence of host DNA. Inhibitors stages in host tissue that are based on nucleic acid present in host DNA have been known to compromise sequences must meet certain requirements to ensure PCR amplification of parasite DNA extracted from reliable and accurate recognition of target DNA. infected host tissue (Lane et al. 1997, Lo et al. 1997), Assays must prove to be: sensitive enough to detect outlining the need to determine concentrations of host low levels of infection and/or individual cells; unable to DNA, if any, that jeopardise the sensitivity of the M. cross react with host tissue; and specific to the targeted sydneyi primer pair. While it is evident that PCR amplification of M. sydneyi is hindered following addition of increasing host DNA concentrations, detection of indi- vidual parasites is possible using this experimental approach with the addition of Fig. 8. Adjacent histological sections of Saccostrea commercialisinfected with sporulating stages of Marteilia sydneyi. (A) In situ hybridisation; (B)H&E stain. Mature sporonts shed into the lumen of the digestive tubules are indicated by the white block arrow. Some of the many groups of mature sporonts within the digestive tilbule walls are indicted by arrowheads. Groups of immature sporonts are depicted by arrows. Ct: connective tissue; Dt: digestive tubule; L:lumen. Scale bar = 50 pm The variable signal intensity and inconsistent de- 1984) which does not variegate as the spore matures tection of mature spores within the oyster digestive (Perkins & Wolf 1976), is an unlikely explanation for tubules introduce some concerns regarding the relia- differences in probe and antibody penetration in bility of the in situ hybridisation procedure for use in immature and mature spores in this study. Concentric life cycle investigations. Mature spores are shed from myelin whorls, composed of degraded extraspore cyto- the oyster host coupled in intact sporonts (Roubal et al. plasm, progressively enclose the spore as it matures 1989) and presumably represent the stage infective to within the sporont (Perkins & Wolf 1976); however, the intermediate host. Personal observations revealed given that fixatives and embedding agents easily that approximately 80% of mature spores, classified penetrate mature spores of M. sydneyi for ultrastruc- according to the size of refringent granules In the tural study and are excluded in preparations of mature extraspore cytoplasm (Roubal et al. 1989),were amen- spores of Haplosporidia (Perkins 1990), the mem- able to hybridisation, alleviating some anxiety. Further- branes should not compromise prohe or an!ihcrly more, spores w~thin sporonts observed within the penetration. lumen of the digestive tubules and intestine manifest Reduced signal intensity in mature spores, localised strong and consistent hybridisation signals. Stokes & within the inner sporoplasms, may be directly related Burreson (1995) and Stokes et al. (1995) reported poor to the proportion of target DNA available. The charac- hybridisation of mature spores of 2 haplosporidian spe- teristic cell-within-a-cell development of the Para- cies, Haplospondiurn nelsoni and Minchinia teredinis, myxea is also associated with successive degeneration and attributed this to the inability of either (or both) the of superseded cells (Desportes 1984, Perkins 1988, probe or anti digoxygenin antibody to penetrate the Desportes & Perkins 1990). Reduction of the outer- thick spore wall. Impermeability of the spore wall in sporal cell to a thin cytoplasmic layer occurs during Marteilia spp., described as a 'thin envelope' (Desportes spore maturation of Pal-amyxa paradoxa, and in ma- Kleeman & Adlard: Molecular detection of Marteilia sydneyi 145 ture spores of Marteilia sydneyi, absence of distinct Anderson TJ. Lester RJG (1992) Sporulation of Marteilioides ribosomes, lysis of plasmalemma and electronlucent branchialis n. sp. (Paramyxea) in the Sydney rock oyster. appearance of ground cytoplasm (Perkins 1991) may Saccostrea commercialis. an electron microscope study. J Protozool 39502-508 explain lack of hybridisation s~gnalin the outermost Anderson TJ, Adlard RD, Lester RJG (1995) Molecular d~ag- sporoplasm of mature spores. Sporonts shed from the nosis of Marteilja sydneyi (Paramyxea) in Sydney rock oyster would represent the most mature spore stages, oysters, Saccostrea commercialis (Angas). J Fish Dis 18: yet their detection was reliable, suggesting that some 507-510 Cabot EL, Beckenbach AT (1989) Simultaneous editing of mature spores in tissue may have an inactive phase or multiple nucleic acid and protein sequences with ESEE. are no longer viable. The combined use of viability Comput Appl Biosci 5:233-234 stains and rRNA probes have been used to differenti- Chang PS, Lo CF, Wang YC, Kou GH (1996) Identification ate viable, inactive and dead cells of bacteria in situ of white spot syndrome associated baculovirus (WSBV) (Williams et al. 1998) and may shed some light on this target organs in the shnmp Penaeus monodon by In situ hybridization. Dis Aquat Org 27:131-139 phenomenon. Desportes 1 (1984) The Paramyxea Levine 1979: an original The specificity of the LEGl/PR02 primer pair and example of evolution towards multicellularity. Origins Life DIG-labelled DNA probe is yet to be tested on other 13:343-352 Paramyxea species. The paramyxean Marteilioides Desportes I,Perkins F0 (1990) Phylum Paramyxea. In: Mar- gulis L, Corliss JO, Melkonian M. Chapman DJ (eds) branchialis infects the gills of Saccostrea commercialis Handbook of Protoctista. Jones and Bartlett Publishers, and is found in QX endemic areas (Anderson & Lester Boston, p 30-35 1992). The likelihood of cross-reaction with this spe- Diggles B, Adlard RD (1995) Taxonomic affinities of Crypto- cies, or other as yet undiscovered species, must be caryon irritans and Ichthyophthirius multifiliis inferred assessed. Given that the interspecific variability in the from ribosomal RNA sequence data. Dis Aquat Org 22: 39-43 nucleotide sequence of internal transcribed spacer Durand S, Lightner DV, Nunan LM, Redman RM, Man J, regions have allowed discrimination between con- Bonami JP. (1996) Application of gene probes as diagnos- generic species (Adlard et al. 1993), probes designed tic tools for white spot baculovirus (WSBV) of penaeid within the given gene sequence may prove to be spe- shrimp. Dis Aquat Org 2759-66 Fong D, Chan MMY, Rodnguez R, Chen CC, Liang Y. Little- cies specific to Marteilia sydneyi. wood DTJ, Ford SE (1993) Small subunit ribosomal RNA The combined use of the PCR and in sltu hybridisa- gene sequence of the parasitic protozoan Haplosporidian tion tests developed and optimised in this study should nelsoni provides a molecular probe for the oyster MSX provide a valuable research tool for the detection and disease. Mol Biochem Parasitol62:139-142 localisation of Marteilia sydneyi life cycle stages in Lane JE, Olivares-Villagomez D, Vnencak-Jones CL. Mc- Curley TL, Carter CE (1997) Detection of Trypanosoma potential alternate hosts. While the high detection cruzi with the polymerase chain reaction and In situ sensitivity of the PCR test will allow rapid screening of hybridization in infected murine cardiac tissue Am J Trop large numbers of potential alternative hosts for the Med Hyg 56588-595 presence of parasite DNA, it does not identify the Lester RJG (1986) Field and laboratory observations on the oyster parasite Marteilia sydneyi. In. Cremin M, Dobson infective stages themselves. In situ hybridisation con- C, Moorhouse DE (eds) Parasite 11ves. Un~versity of ducted on paraffin sections will determine the locality Queensland Press, Brisbane, p 33-40 of the parasite within the host and allow subsequent Li W, Graur D (1991) Fundamentals of molecular evolution. morphological characterisation. Sinauer Associates Inc. Sunderland, MA, p 70-75 Lo CF, Ho CH, Chen CH, Liu KF, Chiu YL. Yeh PY, Peng SE, Hsu HC, LIU HC, Chang CF, Su MS, Wang CH. Kou GH Acknowledgements. We thank Dr Jess Morgan, University of (1997) Detection and tissue tropism of white spot syndrome Queensland, for her constructive suggestions and for pro- baculovirus (WSBV) in captured brooders of Penaeus viding assistance with PCR reactions. This research was monodon with a special emphasis on reproductive organs. supported by the University of Queensland Enabling Grant Dis Aquat Org 30:53-72 (OO9G98) to Professor Robert Lester and R.D.A. Mialhe E, Bachere E, Le Bec C, Grizel H (1985) Isolement et purification de Marteilia (Protozoa: Ascetospora) parasite LITERATURE CITED de bivalves marins. CR Acad Sci (Ser 111) 302:137-142 Perkins F0 (1988) Parasite morphology, strategy, and evolu- Adlard RD, Ernst I (1995) Extended range of the oyster tion. Am Fish Soc Spec Publ 18:93-111 pathogen Martellia sydneyl. Bull Eur Assoc Fish Pathol 15: Perkins F0 (1990) Phylum Haplosporidia. 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Dis Aquat ratory Press, Cold Spring Harbour, NY Org 36221-226 Stokes NA, Burreson EM (1995) A sensitive and specific DNA Williarns SC, Hong Y, Danavall DCA, Howard-Jones MH, probe for the oyster pathogen Haplosporidium nelsoni. G~bsonD, Frischer ME, Verity PG (1998) Distinguishing J Eukaryot Microbiol 42:350-357 between living and non living bacteria: evaluation of the Stokes NA, Siddall ME, Burreson EM (1995) Small subunit vital stain propidium iodide and its combined use with ribosomal RNA gene sequence of Minchinia teredinis molecular probes in aquatic samples. J Microbiol Methods (Haplosporidia: Haplosporidiidae) and a specific DNA 32~225-236 Editorial responsibility: Larry Vaughan, Submitted: January 10, 1999; Accepted: October 24, 1999 Portsmouth, New Hampshire, USA Proofs received from au?hor(s):February 15, 2000