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Small subunit ribosomal RNA gene sequence of the parasitic protozoan Haplosporidium nelsoni provides a molecular probe for the oyster MSX disease
Molecular and Biochemical Parasitology, 62 (1993) 139-142
139
© 1993 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/93/$06.00 MOLBIO 02081
Short C o m m u n i c a t i o n
Small subunit ribosomal RNA gene sequence of the parasitic protozoan Haplosporidium nelsoni provides a molecular probe for the oyster MSX disease Dunne Fong
a •
, M a r i o n M a n - Y i n g C h a n a, R o b e r t o R o d r i g u e z a, C h i a n n - C h y i C h e n a, Y o n g Liang b, D. Tim J. Littlewood b'l and Susan E. F o r d b
aBureau of Biological Research and Department of Biological Sciences, Rutgers, The State University of New Jersey, C226 Nelson Biological Laboratories, Piscataway, NJ 08855-1059, USA; bHaskin Shellfish Research Laboratory, Department of Marine and Coastal Sciences, Rutgers, The State University of New Jersey, Port Norris, NJ 08349, USA (Received 29 July 1993; accepted 7 September 1993)
Key words: Crassostrea virginica; Haplosporidium nelsoni; MSX disease; small subunit r R N A
The protozoan Haplosporidium nelsoni, etiological agent of MSX disease, causes heavy mortalities of the eastern oyster, Crassostrea virginica, on the Atlantic coast of the United States. Although this parasite was first identified in 1957, to date, its complete life cycle, or that of any other Acetosporan protozoan, is still unknown; these protozoa cannot be cultured [1]. The current detection method for H. nelsoni is by examination of stained histological sections of oyster tissues [2]. Thus, a technique to rapidly and sensitively detect this economically important oyster parasite is much needed. As a first step in the development of a molecular probe specific to the parasite, we report the nucleotide sequence of the small subunit (SSU) ribosomal RNA (rRNA) of H. nelsoni and in situ hybridization with cells from infected oyster hemolymph ~-orresponding author. Tel: 908-932-5268; Fax: 908-932-5870.
1Present address: Department of Palaeontology, British Museum (Natural History), London SW7 5BD, UK. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number X74131.
using a fluorescent oligonucleotide probe based on the H. nelsoni rRNA sequence. The SSU rRNA gene of H. nelsoni was obtained by polymerase chain reaction (PCR) and molecular cloning. The parasites were enriched from infected oyster hemolymph by panning [3]. DNA was isolated by proteinase K-phenol extraction and PCR was performed using the universal primers for eukaryotic SSU rRNAs (35 cycles at 92°C for 1 min, 60°C for 2 min and 72°C for 3 min, with Perkin Elmer reagents and Thermolyne TEMP-TRONIC thermocycler) [4]. The PCR products were cloned by the TA technique into the pCRII vector (Invitrogen) and the bacterial host XL1BLUE (Stratagene). Positive clones were characterized by restriction mapping. DNA sequencing was performed using [~-35S]dATP and Sequenase Version 2.0 (US Biochemicals) by the dideoxy chain termination method, using both universal and specific rRNA primers [5]. With the restriction enzyme BamHI, only two patterns of restriction digest were seen among the cloned inserts: either two or three DNA fragments were detected by agarose gel analysis (data not shown). Previously, our
140
1 AACCTGGTTGATCCTGCCAGTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCCAAGTATAAACACGT~TA~~ I00 illillililIllilllilililllilllll111JilllllJlitlllllllJlllJlt iiltl i II I III II IIlllll II illill 1 AACCTC,G T T G A T C C T G C C A G T A G T C A T A T G C T T G T C T C A A A G A T T A A G C C A T G C A T G T C T A A G T A C A T A C . . . T C T T G C A C A G T G A A A C C G C G A A T G G C T 100 I01 C A T T A C A A C A G T T A T A A T T T A T T T G . G A A T G T A A A A T T A C T C G G A T A A C C G T A G T A A . C C T A G G G C T ~ T A C ~ G . A T ~ T ~ A C ~ A ~ ilJDi i lltIltl II Lil I I II IIII lillilll I IIII i}il itilIlSl tl I I ] I lJ 98 C A T T A A A T C A G T T A T G G T T C A T T A G A T G G T A C A A T C C T A C T T G G A T A A C T G T G G T A A T T C T A G A G C T A A T A C A T ~ C ~ C ~ C ~ A C ~
197 I
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198 ACCATCATTGGTTGCATATATAACATTGCTTCCGATCGCGTGGGTTAAA A A C C A G T C ..................................... GTACAA 260 I II II II I i II 1 11 I II t I II II 198 GAGCGC~-F~-~.ATTAGATCAAAACCAATCGGTCGCAAGGCCGTCCGTTTTGGTGACTCTGGATAACTTTGAGCAGATCGCACGGG~TGTCCCGGCGAC~ 297 261 ATCATTCAAGTTTCTGCCCTATCAGC..TTGACGGTAGGATATGGGCCTACCGTG(M:TATGACGGGTAACGGGGAATCAGGGTTCGATTCGGGAGAGGGA II IIill I IltlIlllllll [ I II fill i I IIllllJ III I I lllIllllItllllltliillillIllll lilltllll 298 G T L È ~ z - r C A A A T G T C T G C C C T A T C A A C T G T C G A T G G T A C G T G C T A T G C C T A C C A T G G T T G T A A C G G G T A A C G G G G A A T C A ~ A ~ C ~ A ~ 359 G C C T G A G A A A ~ T A C C A C A T C C A C G G A C G G C A G C A G G T G C G C A A A T T A C C C A C T T C T G A C T C A G A G A G ~ A G ~ C ~ C A T ~ C ~ T . . II iJliIlJillililliJlilll III lllilllll lilllllilJlllll~ Iti I I 1 llIIillllll I i llIll 398 G C A T G A G A A A C G G C T A C C A C A T C C A A G G A A G G C A G C A G G C G C G C A A A T T A C C C A C T C C T G G C A C ~ G G T A G T G A C G A A A A A T ~ C ~ T ~
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555 A C T A G C G T A T A C T A A A G T T G . T G C T G T T A A A A C G C T C G T A G T T G G A T A T C A G T T A T G T G A G C G T G G A C A G G G T G T G T A T A C A ~ G ~ T C C A ~ 653 I IIIIIIIII lililllJ III il[llll illlllll IIIII II I I I I II i 1 I 1 I I 1 598 AATAGCGTATATTAAAGTTGCTGCAGTTAAAAAGCTCGTAGCTGGATCT C G G G T C C A G G C T G G C G G T C c A C C T C G T T G T G G T T A C T G ............ C 685 654 TTGTTGL-"~CACCAAACTTAATTCAGGACTGCCATTCTGACCATTTCTGTGGCTCGGGTTGGCG T G T C C T G A T C T T T T A ~ F F I ' G A ~ T T ~ G ~ IIII IIII II I I II I II ] I II I i II i Ill II I lllllillilll lllilll fill 686 C T G T T C T G A C C C A A C C T C T C G G T T G T A A A C C C T T G G T ~ T G A T T G A G . . . T T G C T T C G G G T G G C C G A A A C T T T T A C T T T C w % A A A A A T T A G A G T G T T C
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1532 G T T G G G A T A G A T G A T T G C A A T T G T T C A T t ` T T G A A C A A G G A A T A T C T A G T A A A C G C A A G T C A T C A A C T T C ~ C A T T G A T T A ~ C ~ T ~ A C ~ 1631 I IIIII I I II I I I I II I IIIII IIIIit I IIIII II1 I Itll I I II I I I I I I I I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1551 C T A G G G A T T C ' G G G T G T G T A A T T ~ A T C C C C A T G A A C G A G G A A T T C C C A G T A A G C G C G A A T C A T A A G T T C G C ~ A ~ A ~ ~ A ~ 1649 P141 1632 CGCCCGTCGCTTCTACCGATTGAATAATACGGTCA~"TC, GGTGGA- ~"F~AdAGACG ........... R ~ T T C G G A T C G T T T C T G C A G A A G C T T C G T G A 1719 IIIIIllllll IIIlllllll II I I1 I I IIII II I III1 III111 I I IIII I I 1650 C C ~ C C C G T C G C T A C T A C C G A T T G C ~ T C ~ t ~ t ~ z ~ A G T G A G C C C C T T G G A C T G ~ T C C C G A C G G C C G G C A A C G C G T T T T C ~ ~ C ~ ~ 1749 1720 ACCTTATTATTTAGAGGAAGGAAAAGTCGTAACAAGGTTACCGTAGGTGAACCTGCAGAAGGATCAA II II I I I I I I I I I IlllllllJllllllltl I IIIIIIIIlllllllltllllllll 1750 ACT~'fACTTTCTAGAGGTCGTAAAAGTCGTAACAAGGTTTCTGTAGGTGAACCTGCAGAAGGATCAA
F i g . 1. C o m p a r i s o n
of small subunit ribosomal RNA sequences of show 72.4% sequence identity. The location
1786
H.nelsoni
1816
C.virginica
Crassostrea virginica a n d Haplosporidium nelsoni. of PI4 oligonucleotide
is m a r k e d .
The two
141
work on the oyster rRNA sequence has shown two BamHI sites, i.e. SSU rRNA of C. virginica can be cut into three fragments by BamHI (accession number X60315) [5]. Thus, the other cloned insert most likely belonged to the parasite, since the original hemolymph samples had been enriched for H. nelsoni and no other microorganism was seen microscopically. From sequencing, the H. nelsoni SSU rRNA is 1786 bp in size; comparison between oyster and parasite rRNA sequences has been made (Fig. 1). The identity of the rRNA sequence of H. nelsoni was confirmed by in situ hybridization. A fluorescein-oligonucleotide was synthesized (designated P14, FITC-5'-TCGTCTCTAACATCCACCCAG-3', by Bio-Synthesis). P14 was selected based on sequence comparison between C. virginica and H. nelsoni, and because the sequence did not appear elsewhere in the oyster SSU rRNA (Fig. 1). Cells from infected hemolymph were attached onto silanecoated glass slides (Oncor), fixed in buffered 10% formalin, treated with trypsinogen and alcohol, and air-dried [6]. A sodium borohydride step was performed to decrease autofluorescence [7]. In situ hybridization was done in an assay buffer containing 50 mM KCI/ 10 mM Tris-HC1, pH 9 / 0 . 1 % Triton X-100/4.5 mM MgC12, together with 4 #M P14 oligonu-
cleotide, and denatured salmon sperm DNA at 500 /~g ml-1 [8]. After the slide preparations had been overlaid with plastic cover slips, sealed with nail polish and covered with mineral oil, in situ hybridization was performed under PCR conditions in a thermocycler (using the hot start protocol, with 25 cycles at 94°C for 1 min and 55°C for 2 min) [6]. We obtained strong hybridization signals when cells were treated by this procedure, probably due to enhanced oligonucleotide entry and increased rRNA denaturation from the ribosomes. The slides were rinsed in xylene and alcohol, and washed in the assay buffer at 50°C before observation for fluorescence [6]. The fluorescein-oligonucleotide P14 clearly reacted with H. nelsoni and did not bind oyster cells (Fig. 2). In some hemolymph samples where other protozoa were seen, the oligonucleotide did not bind the other cells (data not shown). Thus, the in situ hybridization results confirmed the PCR-TA cloning of rRNA gene sequence of H. nelsoni. To our knowledge, the H. nelsoni SSU rRNA sequence presented here is the first sequence from any Acetosporan protozoan reported. With the availability of this sequence, we have begun to develop molecular probes to detect MSX disease in oysters. The probes may also be useful to investigate the life
Fig. 2. In situ hybridization of cells from infected oyster hemolymph. (A) Phase contrast photomicrograph showing Haplosporidium nelsoni plasmodium (arrow) and oyster hemocytes (all other cells). (B) Same field showing fluoresceinoligonucleotide P14 label. Scale bar = 10 #m.
142
cycle of this parasite. Preliminary study of the H. nelsoni phylogenetic relationship based on SSU rRNA is inconclusive, although the H. nelsoni rRNA sequence is different from that of the apicomplexan Perkinsus sp. [9], members of which are also widespread parasites of oysters. We plan to continue phylogenetic comparisons by analyzing the SSU rRNA sequence from another Acetosporan parasite, such as Haplosporidium costale (from oysters) or Minchinia teredinis (from shipworms) [10], so that there will be two Acetosporan sequences for comparison. Currently, with regard to H. nelsoni, at least one other research group (colleagues at the Virginia Institute of Marine Science) has the same aim as ours (Stokes, N.A. and Burreson, E.M. (1993) Comparison of 16S-like rDNA of Crassostrea virginica and Haplosporidium nelsoni. J. Shellfish Res. 12, 156-157, abstract).
Acknowledgements We thank Dr. Patricia O. Wainright for comments, and K. Ashton-Alcox and R. D. Barber for technical assistance. This work was supported by USDA grant 89-34116-4553 and NOAA Sea Grant NA89AA-D-SG057 (Project R/F-32). This is New Jersey Sea Grant Publication No. NJSG-93-271, Contribution No. 93-22 from the Institute of Marine and Coastal Sciences at Rutgers University, and New Jersey Agricultural Experiment Station Publication No. D-32901-1-93, supported by state funds.
References 1 Haskin, H.H. and Andrews, J.D. (1988) Uncertainties and speculations about the life cycle of the eastern oyster pathogen Haplosporidium nelsoni (MSX). In: Disease Processes in Marine Bivalve Molluscs (Fisher, W.S., ed.), pp. 5~2, American Fisheries Society Special Publications 18, Bethesda. 2 Ford, S.E. and Tripp, M.R. (In Press) Diseases and defense mechanisms of the eastern oyster Crassostrea virginica. In: The Eastern Oyster Crassostrea virginica (Eble, A.F., Kennedy, V.S. and Newell, R.1.E., eds.), Maryland Sea Grant, College Park. 3 Ford, S.E., Kanaley, S.A., Ferris, M. and AshtonAlcox, K.A. (1990) 'Panning', a technique for enrichment of the parasite Haplosporidium nelsoni (MSX) from hemolymph of infected oysters. J. Invert. Pathol. 56, 347 352. 4 Medlin, L., Elwood, H.J., Stickel, S. and Sogin, M.L. (1988) The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71, 491-499. 5 Littlewood, D.T.J., Ford, S.E. and Fong, D. (1991) Small subunit rRNA gene sequence of Crassostrea virginica (Gmelin) and a comparison with similar sequences from other bivalve molluscs. Nucleic Acids Res. 19, 6048. 6 Nuovo, G.J. (1992) PCR in situ Hybridization, Raven, New York. 7 DeLong, E.F., Wickham, G.S. and Pace, N.R. (1989) Phylogenetic stains: ribosomal RNA-based probes for the idenification of single cells. Science 243, 1360-1363. 8 Baldino, F., Jr., Robbins, E. and Lewis, M.E. (1992) Detection of mRNA in situ with DIG-labeled synthetic oligonucleotide probes. In: Nonradioactive Labeling and Detection of Biomolecules (Kessler, C., ed.), pp. 367-373, Springer-Verlag, Berlin. 9 Goggin, C.L. and Barker, S.C. (1993) Phylogenetic position of the genus Perkinsus (Protista, Apicomplexa) based on small subunit ribosomal RNA. Mol. Biochem. Parasitol. 60, 65 70. 10 Perkins, F.O. (1990) Phylum Haplosporidia. In: Handbook of Protoctista (Margulis, L., Corliss, J.O., Melkonian, M. and Chapman, D.J., eds.), pp. 1%29, Jones and Bartlett, Boston.
139
© 1993 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/93/$06.00 MOLBIO 02081
Short C o m m u n i c a t i o n
Small subunit ribosomal RNA gene sequence of the parasitic protozoan Haplosporidium nelsoni provides a molecular probe for the oyster MSX disease Dunne Fong
a •
, M a r i o n M a n - Y i n g C h a n a, R o b e r t o R o d r i g u e z a, C h i a n n - C h y i C h e n a, Y o n g Liang b, D. Tim J. Littlewood b'l and Susan E. F o r d b
aBureau of Biological Research and Department of Biological Sciences, Rutgers, The State University of New Jersey, C226 Nelson Biological Laboratories, Piscataway, NJ 08855-1059, USA; bHaskin Shellfish Research Laboratory, Department of Marine and Coastal Sciences, Rutgers, The State University of New Jersey, Port Norris, NJ 08349, USA (Received 29 July 1993; accepted 7 September 1993)
Key words: Crassostrea virginica; Haplosporidium nelsoni; MSX disease; small subunit r R N A
The protozoan Haplosporidium nelsoni, etiological agent of MSX disease, causes heavy mortalities of the eastern oyster, Crassostrea virginica, on the Atlantic coast of the United States. Although this parasite was first identified in 1957, to date, its complete life cycle, or that of any other Acetosporan protozoan, is still unknown; these protozoa cannot be cultured [1]. The current detection method for H. nelsoni is by examination of stained histological sections of oyster tissues [2]. Thus, a technique to rapidly and sensitively detect this economically important oyster parasite is much needed. As a first step in the development of a molecular probe specific to the parasite, we report the nucleotide sequence of the small subunit (SSU) ribosomal RNA (rRNA) of H. nelsoni and in situ hybridization with cells from infected oyster hemolymph ~-orresponding author. Tel: 908-932-5268; Fax: 908-932-5870.
1Present address: Department of Palaeontology, British Museum (Natural History), London SW7 5BD, UK. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number X74131.
using a fluorescent oligonucleotide probe based on the H. nelsoni rRNA sequence. The SSU rRNA gene of H. nelsoni was obtained by polymerase chain reaction (PCR) and molecular cloning. The parasites were enriched from infected oyster hemolymph by panning [3]. DNA was isolated by proteinase K-phenol extraction and PCR was performed using the universal primers for eukaryotic SSU rRNAs (35 cycles at 92°C for 1 min, 60°C for 2 min and 72°C for 3 min, with Perkin Elmer reagents and Thermolyne TEMP-TRONIC thermocycler) [4]. The PCR products were cloned by the TA technique into the pCRII vector (Invitrogen) and the bacterial host XL1BLUE (Stratagene). Positive clones were characterized by restriction mapping. DNA sequencing was performed using [~-35S]dATP and Sequenase Version 2.0 (US Biochemicals) by the dideoxy chain termination method, using both universal and specific rRNA primers [5]. With the restriction enzyme BamHI, only two patterns of restriction digest were seen among the cloned inserts: either two or three DNA fragments were detected by agarose gel analysis (data not shown). Previously, our
140
1 AACCTGGTTGATCCTGCCAGTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCCAAGTATAAACACGT~TA~~ I00 illillililIllilllilililllilllll111JilllllJlitlllllllJlllJlt iiltl i II I III II IIlllll II illill 1 AACCTC,G T T G A T C C T G C C A G T A G T C A T A T G C T T G T C T C A A A G A T T A A G C C A T G C A T G T C T A A G T A C A T A C . . . T C T T G C A C A G T G A A A C C G C G A A T G G C T 100 I01 C A T T A C A A C A G T T A T A A T T T A T T T G . G A A T G T A A A A T T A C T C G G A T A A C C G T A G T A A . C C T A G G G C T ~ T A C ~ G . A T ~ T ~ A C ~ A ~ ilJDi i lltIltl II Lil I I II IIII lillilll I IIII i}il itilIlSl tl I I ] I lJ 98 C A T T A A A T C A G T T A T G G T T C A T T A G A T G G T A C A A T C C T A C T T G G A T A A C T G T G G T A A T T C T A G A G C T A A T A C A T ~ C ~ C ~ C ~ A C ~
197 I
II 197
198 ACCATCATTGGTTGCATATATAACATTGCTTCCGATCGCGTGGGTTAAA A A C C A G T C ..................................... GTACAA 260 I II II II I i II 1 11 I II t I II II 198 GAGCGC~-F~-~.ATTAGATCAAAACCAATCGGTCGCAAGGCCGTCCGTTTTGGTGACTCTGGATAACTTTGAGCAGATCGCACGGG~TGTCCCGGCGAC~ 297 261 ATCATTCAAGTTTCTGCCCTATCAGC..TTGACGGTAGGATATGGGCCTACCGTG(M:TATGACGGGTAACGGGGAATCAGGGTTCGATTCGGGAGAGGGA II IIill I IltlIlllllll [ I II fill i I IIllllJ III I I lllIllllItllllltliillillIllll lilltllll 298 G T L È ~ z - r C A A A T G T C T G C C C T A T C A A C T G T C G A T G G T A C G T G C T A T G C C T A C C A T G G T T G T A A C G G G T A A C G G G G A A T C A ~ A ~ C ~ A ~ 359 G C C T G A G A A A ~ T A C C A C A T C C A C G G A C G G C A G C A G G T G C G C A A A T T A C C C A C T T C T G A C T C A G A G A G ~ A G ~ C ~ C A T ~ C ~ T . . II iJliIlJillililliJlilll III lllilllll lilllllilJlllll~ Iti I I 1 llIIillllll I i llIll 398 G C A T G A G A A A C G G C T A C C A C A T C C A A G G A A G G C A G C A G G C G C G C A A A T T A C C C A C T C C T G G C A C ~ G G T A G T G A C G A A A A A T ~ C ~ T ~
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456 C C T A T C G T G T G C C T A G T C G T A A T G A G A A C A A T T T A A A A T C A T T A T C G A C T A A C A A G T G G A ~ ~ G C . A G C C ~ T A C C A ~ C I I I I II I 1 IIIIII III lllIil 1 III IIII I I illlIllilllllllllllilIl llllillllll 498 C T T C G A G G C C C C G T A A T T G G A A T G A G T A C A C t Y ~ ? A A A T C C T T T A G C G A G G A T C C A T T G C ~ A G G G C ~ ~ C C A ~ G A G C C ~ C A ~
358 397
.GTC,CAG 455 I I 497 554 illlIlll 597
555 A C T A G C G T A T A C T A A A G T T G . T G C T G T T A A A A C G C T C G T A G T T G G A T A T C A G T T A T G T G A G C G T G G A C A G G G T G T G T A T A C A ~ G ~ T C C A ~ 653 I IIIIIIIII lililllJ III il[llll illlllll IIIII II I I I I II i 1 I 1 I I 1 598 AATAGCGTATATTAAAGTTGCTGCAGTTAAAAAGCTCGTAGCTGGATCT C G G G T C C A G G C T G G C G G T C c A C C T C G T T G T G G T T A C T G ............ C 685 654 TTGTTGL-"~CACCAAACTTAATTCAGGACTGCCATTCTGACCATTTCTGTGGCTCGGGTTGGCG T G T C C T G A T C T T T T A ~ F F I ' G A ~ T T ~ G ~ IIII IIII II I I II I II ] I II I i II i Ill II I lllllillilll lllilll fill 686 C T G T T C T G A C C C A A C C T C T C G G T T G T A A A C C C T T G G T ~ T G A T T G A G . . . T T G C T T C G G G T G G C C G A A A C T T T T A C T T T C w % A A A A A T T A G A G T G T T C
753 II 782
754 AAAGCAGGC.TTTCGCCTGAATGCAT.TAGCATGGAATAATAAAACACGA~"~-F~GGCATTAGGTTTCAGACCTAAGTGTTTTATTC~GTTTTTGGGCCA..
IIIIIIIII
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850 A A G T A A T G A T T G A T ~ A C ~ G C T A G T A T C A T C G G G T T A G A G G T T A A A T T C T A T G A C C C C G G T G A G A C T G A C T T A ~ A ~ A ~ I IIIIIIIII i III II llill I il i lillllllll III i] II II I IIII I Jl 866 A G G T A A T G A T T A A G A G G G A C T G A C ~ A T T C G T A T G G C ~ T A G A G G T G A A A ~ G G A T C C T C G C C A G A C G G C C T A C A G C G A A A ~ A ~
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1049 I I 1065
1050 AGTTTACTTC ......... C T C A G A A C T T T G A G A G A A A T C A A A G T T T T C G G A C T C A G G G G G G A G T A T G C T C G C A A G G G T G A A A C T T G A A G A A A ~ C ~
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1141 AACGGCACCACCAGATGTGGAGCCTGCGC, CTTAATTTGACTCAACAC. . . T A A A C T T A C C A G G A C C A G A C A T A G T A A G G A T T G A C A G A T T C A . A G T T C T T
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1166 A A ~ A C C A C C A G G A G T G G A G C C T G C . G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C C G G C C C G A A C A C T G T A A G G A T T G A C A G A T T G A G A G C T ~
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1237 T C T T G A T T C . A T G C A T A G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G C G A T T T G T C T G G T T A A T T C C G T T A A C G A A C G A G A C C T C A G C C A T C T A A ~ A ~ IIIit1111 II t IIIIIIIIIIIIit1111t IIIII IIIIIIIIIit11111111111111 t111111111111 I II 1111 I I I 1265 '~-.~,'~C~T~'I~ATC~C~TC~"rAG~I~,C~.GCG~~C~ ~ a ~ a ~ ~ & ~ A ~
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1336 ~ ' ~ ' C C ~ ' ~ . ~ . ~ ' I ~ G G ~ T ~ C G T C ~ - ~ , ~ A ~ A ~ T ~ ~ T ~ T ~ A ~ ~ a ~ ~ ~ A ~ I I I I I I 1 II IIIII IIIIIII I I I I II I I II IIIIIIIIIIIII IIIIII 1365 TCACCGATCTATGCTGTCGG ........... TGCAACTTCTTAGAGGG A C A A G T G G C G T T T A G C C A C A C G . . . A G A T T G A G C A A T A A C A G G T C T G T G A T G
1435
1436 CCCTTAGATGCTCTGGC~TGCACGCGCGCTACAATGTTGCACGTAAC..GAGTTCAACCTTGCCTGAGAAGGCCGGGTAATC..TCC,
1531
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1451 C C C T T A G A T G T T C G G G G C C G C A ~ G C G C G C T A C A C T G A A G G A A T C A A C A G G T A T C T G T C C T G G C C C G A A A ~ ~ C C ~ C ~
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1532 G T T G G G A T A G A T G A T T G C A A T T G T T C A T t ` T T G A A C A A G G A A T A T C T A G T A A A C G C A A G T C A T C A A C T T C ~ C A T T G A T T A ~ C ~ T ~ A C ~ 1631 I IIIII I I II I I I I II I IIIII IIIIit I IIIII II1 I Itll I I II I I I I I I I I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1551 C T A G G G A T T C ' G G G T G T G T A A T T ~ A T C C C C A T G A A C G A G G A A T T C C C A G T A A G C G C G A A T C A T A A G T T C G C ~ A ~ A ~ ~ A ~ 1649 P141 1632 CGCCCGTCGCTTCTACCGATTGAATAATACGGTCA~"TC, GGTGGA- ~"F~AdAGACG ........... R ~ T T C G G A T C G T T T C T G C A G A A G C T T C G T G A 1719 IIIIIllllll IIIlllllll II I I1 I I IIII II I III1 III111 I I IIII I I 1650 C C ~ C C C G T C G C T A C T A C C G A T T G C ~ T C ~ t ~ t ~ z ~ A G T G A G C C C C T T G G A C T G ~ T C C C G A C G G C C G G C A A C G C G T T T T C ~ ~ C ~ ~ 1749 1720 ACCTTATTATTTAGAGGAAGGAAAAGTCGTAACAAGGTTACCGTAGGTGAACCTGCAGAAGGATCAA II II I I I I I I I I I IlllllllJllllllltl I IIIIIIIIlllllllltllllllll 1750 ACT~'fACTTTCTAGAGGTCGTAAAAGTCGTAACAAGGTTTCTGTAGGTGAACCTGCAGAAGGATCAA
F i g . 1. C o m p a r i s o n
of small subunit ribosomal RNA sequences of show 72.4% sequence identity. The location
1786
H.nelsoni
1816
C.virginica
Crassostrea virginica a n d Haplosporidium nelsoni. of PI4 oligonucleotide
is m a r k e d .
The two
141
work on the oyster rRNA sequence has shown two BamHI sites, i.e. SSU rRNA of C. virginica can be cut into three fragments by BamHI (accession number X60315) [5]. Thus, the other cloned insert most likely belonged to the parasite, since the original hemolymph samples had been enriched for H. nelsoni and no other microorganism was seen microscopically. From sequencing, the H. nelsoni SSU rRNA is 1786 bp in size; comparison between oyster and parasite rRNA sequences has been made (Fig. 1). The identity of the rRNA sequence of H. nelsoni was confirmed by in situ hybridization. A fluorescein-oligonucleotide was synthesized (designated P14, FITC-5'-TCGTCTCTAACATCCACCCAG-3', by Bio-Synthesis). P14 was selected based on sequence comparison between C. virginica and H. nelsoni, and because the sequence did not appear elsewhere in the oyster SSU rRNA (Fig. 1). Cells from infected hemolymph were attached onto silanecoated glass slides (Oncor), fixed in buffered 10% formalin, treated with trypsinogen and alcohol, and air-dried [6]. A sodium borohydride step was performed to decrease autofluorescence [7]. In situ hybridization was done in an assay buffer containing 50 mM KCI/ 10 mM Tris-HC1, pH 9 / 0 . 1 % Triton X-100/4.5 mM MgC12, together with 4 #M P14 oligonu-
cleotide, and denatured salmon sperm DNA at 500 /~g ml-1 [8]. After the slide preparations had been overlaid with plastic cover slips, sealed with nail polish and covered with mineral oil, in situ hybridization was performed under PCR conditions in a thermocycler (using the hot start protocol, with 25 cycles at 94°C for 1 min and 55°C for 2 min) [6]. We obtained strong hybridization signals when cells were treated by this procedure, probably due to enhanced oligonucleotide entry and increased rRNA denaturation from the ribosomes. The slides were rinsed in xylene and alcohol, and washed in the assay buffer at 50°C before observation for fluorescence [6]. The fluorescein-oligonucleotide P14 clearly reacted with H. nelsoni and did not bind oyster cells (Fig. 2). In some hemolymph samples where other protozoa were seen, the oligonucleotide did not bind the other cells (data not shown). Thus, the in situ hybridization results confirmed the PCR-TA cloning of rRNA gene sequence of H. nelsoni. To our knowledge, the H. nelsoni SSU rRNA sequence presented here is the first sequence from any Acetosporan protozoan reported. With the availability of this sequence, we have begun to develop molecular probes to detect MSX disease in oysters. The probes may also be useful to investigate the life
Fig. 2. In situ hybridization of cells from infected oyster hemolymph. (A) Phase contrast photomicrograph showing Haplosporidium nelsoni plasmodium (arrow) and oyster hemocytes (all other cells). (B) Same field showing fluoresceinoligonucleotide P14 label. Scale bar = 10 #m.
142
cycle of this parasite. Preliminary study of the H. nelsoni phylogenetic relationship based on SSU rRNA is inconclusive, although the H. nelsoni rRNA sequence is different from that of the apicomplexan Perkinsus sp. [9], members of which are also widespread parasites of oysters. We plan to continue phylogenetic comparisons by analyzing the SSU rRNA sequence from another Acetosporan parasite, such as Haplosporidium costale (from oysters) or Minchinia teredinis (from shipworms) [10], so that there will be two Acetosporan sequences for comparison. Currently, with regard to H. nelsoni, at least one other research group (colleagues at the Virginia Institute of Marine Science) has the same aim as ours (Stokes, N.A. and Burreson, E.M. (1993) Comparison of 16S-like rDNA of Crassostrea virginica and Haplosporidium nelsoni. J. Shellfish Res. 12, 156-157, abstract).
Acknowledgements We thank Dr. Patricia O. Wainright for comments, and K. Ashton-Alcox and R. D. Barber for technical assistance. This work was supported by USDA grant 89-34116-4553 and NOAA Sea Grant NA89AA-D-SG057 (Project R/F-32). This is New Jersey Sea Grant Publication No. NJSG-93-271, Contribution No. 93-22 from the Institute of Marine and Coastal Sciences at Rutgers University, and New Jersey Agricultural Experiment Station Publication No. D-32901-1-93, supported by state funds.
References 1 Haskin, H.H. and Andrews, J.D. (1988) Uncertainties and speculations about the life cycle of the eastern oyster pathogen Haplosporidium nelsoni (MSX). In: Disease Processes in Marine Bivalve Molluscs (Fisher, W.S., ed.), pp. 5~2, American Fisheries Society Special Publications 18, Bethesda. 2 Ford, S.E. and Tripp, M.R. (In Press) Diseases and defense mechanisms of the eastern oyster Crassostrea virginica. In: The Eastern Oyster Crassostrea virginica (Eble, A.F., Kennedy, V.S. and Newell, R.1.E., eds.), Maryland Sea Grant, College Park. 3 Ford, S.E., Kanaley, S.A., Ferris, M. and AshtonAlcox, K.A. (1990) 'Panning', a technique for enrichment of the parasite Haplosporidium nelsoni (MSX) from hemolymph of infected oysters. J. Invert. Pathol. 56, 347 352. 4 Medlin, L., Elwood, H.J., Stickel, S. and Sogin, M.L. (1988) The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71, 491-499. 5 Littlewood, D.T.J., Ford, S.E. and Fong, D. (1991) Small subunit rRNA gene sequence of Crassostrea virginica (Gmelin) and a comparison with similar sequences from other bivalve molluscs. Nucleic Acids Res. 19, 6048. 6 Nuovo, G.J. (1992) PCR in situ Hybridization, Raven, New York. 7 DeLong, E.F., Wickham, G.S. and Pace, N.R. (1989) Phylogenetic stains: ribosomal RNA-based probes for the idenification of single cells. Science 243, 1360-1363. 8 Baldino, F., Jr., Robbins, E. and Lewis, M.E. (1992) Detection of mRNA in situ with DIG-labeled synthetic oligonucleotide probes. In: Nonradioactive Labeling and Detection of Biomolecules (Kessler, C., ed.), pp. 367-373, Springer-Verlag, Berlin. 9 Goggin, C.L. and Barker, S.C. (1993) Phylogenetic position of the genus Perkinsus (Protista, Apicomplexa) based on small subunit ribosomal RNA. Mol. Biochem. Parasitol. 60, 65 70. 10 Perkins, F.O. (1990) Phylum Haplosporidia. In: Handbook of Protoctista (Margulis, L., Corliss, J.O., Melkonian, M. and Chapman, D.J., eds.), pp. 1%29, Jones and Bartlett, Boston.