Skip to main content
SpringerLink
Log in

Specific Detection of Pacific Oyster (Crassostrea gigas) Larvae in Plankton Samples Using Nested Polymerase Chain Reaction

  • Short Communication
  • Published:
Marine Biotechnology Aims and scope Submit manuscript

Abstract

Management of sustainable Pacific oyster fisheries would be assisted by an early, rapid, and accurate means of detecting their planktonic larvae. Reported here is an approach, based on polymerase chain reaction (PCR), for the detection of Pacific oyster larvae in plankton samples. Species-specific primers were designed by comparing partial mitochondrial cytochrome oxidase subunit I (COI) sequences from Crassostrea gigas, with other members of the family Ostreidae including those of Crassostrea angulata. Assay specificity was empirically validated through screening DNA samples obtained from several species of oysters. The assay was specific as only C. gigas samples returned PCR-positive results. A nested PCR approach could consistently detect 5 or more D-hinge-stage larvae spiked into a background of about 146 mg of plankton. The assay does not require prior sorting of larvae. We conclude that the assay could be used to screen environmental and ballast water samples, although further specificity testing against local bivalve species is recommended in new locations.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2

Similar content being viewed by others

References

  • InstitutionalAuthorNameABARE. (2004) Australian Fisheries Statistics 2003 ABARE Canberra, Australia 1–58

    Google Scholar 

  • T.J. Anderson R.D. Adlard (1994) ArticleTitleNucleotide-sequence of a rDNA internal transcribed spacer supports synonym of Saccostrea commercialis and S. glomerata J Mollus Stud 60 196–197

    Google Scholar 

  • M.A. Banks D. Hedgecock C. Waters (1993) ArticleTitleDiscrimination between closely related Pacific oyster species (Crassostrea) via mitochondrial DNA sequences coding for large subunit rRNA Mol Mar Biol Biotechnol 2 129–136 Occurrence Handle1:CAS:528:DyaK2cXktVaitr0%3D Occurrence Handle8103411

    CAS  PubMed  Google Scholar 

  • J.L. Bell J.P. Grassle (1998) ArticleTitleA DNA probe for identification of larvae of the commercial surfclam (Spisula solidissima) Mol Mar Biol Biotechnol 7 127–137 Occurrence Handle1:CAS:528:DyaK1cXjslCrsr8%3D Occurrence Handle9628008

    CAS  PubMed  Google Scholar 

  • C.D.K. Bottema G. Sarkar J.D. Cassady S. Ii C.M. Dutton S.S. Sommer (1993) ArticleTitlePolymerase chain reaction amplification of specific alleles: a general method of detection of mutations, polymorphisms, and haplotypes Methods Enzymal 218 388–402 Occurrence Handle1:CAS:528:DyaK3sXltV2qs74%3D Occurrence Handle10.1016/0076-6879(93)18031-7

    Article  CAS  Google Scholar 

  • P. Boudry S. Heurtebise S. Lapègue (2003) ArticleTitleMitochondrial and nuclear DNA sequence variation of presumed Crassostrea gigas and Crassostrea angulata specimens: a new oyster species in Hong Kong? Aquaculture 228 15–25 Occurrence Handle10.1016/S0044-8486(03)00443-5 Occurrence Handle1:CAS:528:DC%2BD3sXosVKqsL4%3D

    Article  CAS  Google Scholar 

  • K.K. Chew (1990) ArticleTitleGlobal bivalve shellfish introductions World Aquacult 21 9–21

    Google Scholar 

  • B.E. Deagle N. Bax C.L. Hewitt J.G. Patil (2003) ArticleTitleDevelopment and evaluation of a PCR-based test for detection of Asterias (Echinodermata : Asteroidea) larvae in Australian plankton samples from ballast water Mar Freshwater Res 54 709–719 Occurrence Handle10.1071/MF03031 Occurrence Handle1:CAS:528:DC%2BD3sXovVGmur8%3D

    Article  CAS  Google Scholar 

  • O. Folmer M. Black W. Hoeh R. Lutz R. Vrijenhoek (1994) ArticleTitleDNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates Mol Mar Biol Biotechnol 3 294–299 Occurrence Handle1:CAS:528:DyaK2MXjt12gtLs%3D Occurrence Handle7881515

    CAS  PubMed  Google Scholar 

  • M.E. Frischer J.M. Danforth L.C. Tyner J.R. Leverone D.C. Marelli W.S. Arnold N.J. Blake (2000) ArticleTitleDevelopment of an Argopecten-specific 18S rRNA targeted genetic probe Mar Biotechnol 2 11–20 Occurrence Handle1:CAS:528:DC%2BD3cXhs1Wqsrs%3D Occurrence Handle10804235

    CAS  PubMed  Google Scholar 

  • M.E. Frischer A.S. Hansen J.A. Wyllie J. Wimbush J. Murray S.A. Nierzwicki-Bauer (2002) ArticleTitleSpecific amplification of the 18S rRNA gene as a method to detect zebra mussel (Dreissena polymorpha) larvae in plankton samples Hydrobiol 487 33–44 Occurrence Handle10.1023/A:1022971918673 Occurrence Handle1:CAS:528:DC%2BD3sXit1Cns7Y%3D

    Article  CAS  Google Scholar 

  • P.M. Gaffney D. Hedgecock A. Robinson (1998) ArticleTitleIdentifying the Kumamoto oyster: a first step in broodstock restoration and improvement J Shellfish Res 17 1270

    Google Scholar 

  • A. Godhe S.K. Otta A.S. Rehnstam-Holm I. Karunasagar I. Karunasagar (2001) ArticleTitlePolymerase chain reaction in detection of Gymnodinium mikimotoi and Alexandrium minutum in field samples from southwest India Mar Biotechnol 3 152–162 Occurrence Handle10.1007/s101260000052 Occurrence Handle1:CAS:528:DC%2BD3MXksVGmsr4%3D Occurrence Handle14961378

    Article  CAS  PubMed  Google Scholar 

  • M.P. Hare S.R. Palumbi C.A. Butman (2000) ArticleTitleSingle-step species identification of bivalve larvae using multiplex polymerase chain reaction Mar Biol 137 953–961 Occurrence Handle10.1007/s002270000402 Occurrence Handle1:CAS:528:DC%2BD3MXkvFWlsw%3D%3D

    Article  CAS  Google Scholar 

  • D. Hedgecock G. Li M.A. Banks Z. Kain (1999) ArticleTitleOccurrence of the Kumamoto oyster Crassostrea sikamea in the Ariake Sea, Japan Mar Biol 133 65–68 Occurrence Handle10.1007/s002270050443

    Article  Google Scholar 

  • J.E. Holliday J.A. Nell (1985) ArticleTitleConcern over Pacific oyster in Port Stephens Aust Fish 44 29–31

    Google Scholar 

  • E. Kenchington C.J. Bird J. Osborne M. Reith (2002) ArticleTitleNovel repeat elements in the nuclear ribosomal RNA operon of the flat oyster Ostrea edulis C. Linneaus, 1758 and O. angasi Sowerby, 1871 J Shellfish Res 21 697–705

    Google Scholar 

  • P. Korringa (1976) Farming the Pacific oyster (Crassostrea gigas) in Hiroshima Bay, Japan Farming the Cupped oyster of the Genus Crassostrea Elsevier Amsterdam, Netherlands 153–182

    Google Scholar 

  • S. Lapègue I. Boutet A. Leitao S. Heurtebise P. Garcia C. Thiriot-Quiévreux P. Boudry (2002) ArticleTitleTrans-Atlantic distribution of a mangrove oyster species revealed by 16S mtDNA and karyological analyses Biol Bull 202 232–242 Occurrence Handle12086994

    PubMed  Google Scholar 

  • W. Menzel (1991) Estuarine and Marine Bivalve Mollusk Culture CRC Press Boca Raton Fla.

    Google Scholar 

  • R. Miserez T. Pilloud X. Cheng J. Nicolet C. Griot J. Frey (1997) ArticleTitleDevelopment of a sensitive nested PCR method for the specific detection of Mycoplasma mycoides subsp. mycoides SC Mol Cell Probe 11 103–111 Occurrence Handle10.1006/mcpr.1996.0088 Occurrence Handle1:CAS:528:DyaK2sXjtFWqu78%3D

    Article  CAS  Google Scholar 

  • Mitchell I., Jones A., Crawford C. (2000). Distribution of feral Pacific oysters and environmental conditions. In: Natural Heritage Trust Final Report. Tasmanian Aquaculture and Fisheries Institute, University of Tasmania

  • B. Morton K. Lam S. Slack-Smith (2003) ArticleTitleFirst report of the European flat oyster Ostrea edulis, identified genetically, from Oyster Harbour, Albany, south-western Western Australia Molluscan Res 23 199–208 Occurrence Handle10.1071/MR03005

    Article  Google Scholar 

  • C.R. Newton A. Graham L.E. Heptinstall S.J. Powell C. Summers N. Kalsheker J.C. Smith A.F. Markham (1989) ArticleTitleAnalysis of any point mutation in DNA: the amplification refractory mutation system (ARMS) Nucleic Acids Res 17 2503–2516 Occurrence Handle1:CAS:528:DyaL1MXktFyktbs%3D Occurrence Handle2785681

    CAS  PubMed  Google Scholar 

  • Ó D. Foighil P.M. Gaffney T.J. Hilbish (1995) ArticleTitleDifferences in mitochondrial 16S ribosomal gene sequences allow discrimination among American [Crassostrea virginica (Gmelin)] and Asian [C. gigas (Thunberg) C. ariakensis Wakiya] oyster species J Exp Mar Biol Ecol 192 211–220 Occurrence Handle10.1016/0022-0981(95)00065-Y Occurrence Handle1:CAS:528:DyaK28Xjt1Chuw%3D%3D

    Article  CAS  Google Scholar 

  • Ó D. Foighil P.M. Gaffney A.E. Wilbur T.J. Hilbish (1998) ArticleTitleMitochondrial cytochrome oxidase I gene sequences support an Asian origin for the Portuguese oyster Crassostrea angulata Mar Biol 131 497–503 Occurrence Handle10.1007/s002270050341 Occurrence Handle1:CAS:528:DyaK1cXltFWju7w%3D

    Article  CAS  Google Scholar 

  • Ó D. Foighil B.A. Marshall T.J. Hilbish M.A. Pino (1999) ArticleTitleTrans-Pacific range extension by rafting is inferred for the flat oyster Ostrea chilensis Biol Bull 196 122–126 Occurrence Handle1:STN:280:DyaK1M3kt12nsg%3D%3D Occurrence Handle10226353

    CAS  PubMed  Google Scholar 

  • J.G. Patil K.R. Hayes R.M. Gunasekera B.E. Deagle F. McEnnulty N. Bax C.L. Hewitt (2004) Port of Hastings National Demonstration Project — Verification of the Type II error rate of the Ballast Water Decision Support System (DSS). Final report prepared for Environmental Protection Authority, CSIRO Marine Research Hobart, Australia

    Google Scholar 

  • Patil, J.G., Gunasekera, R.M., Deagle, B.E., Bax, N., Blackburn, S.J. (in press) “Development and evaluation of a PCR based assay for detection of the toxic dinoflagellate, Gymnodinium catenatum in ballast water and environmental samples.” Biol E Innovations.

  • A. Rocha-Olivares (1998) ArticleTitleMultipex haplotype-specific PCR: a new approach for species identification of the early life stages of rockfishes of the species-rich genus Sebastes Cuvier J Exp Mar Biol Ecol 231 279–290 Occurrence Handle10.1016/S0022-0981(98)00098-7 Occurrence Handle1:CAS:528:DyaK1cXns1Ojt7c%3D

    Article  CAS  Google Scholar 

  • W. Rychlik (1996) OLIGO, Version 5.0 for Macintosh (computer software) National Biosciences Plymouth, Minn.

    Google Scholar 

  • G. Shatkin S.E. Shumway R. Hawes (1997) ArticleTitleConsiderations regarding the possible introduction of the Pacific oyster (Crassostrea gigas) to the Gulf of Maine: a review of global experience J Shellfish Res 16 463–477

    Google Scholar 

  • P.J. Smith H. Ozaki Y. Fujio (1986) ArticleTitleNo evidence for reduced genetic variation in the accidentally introduced oyster Crassostrea gigas in New Zealand N Z J Mar Freshwater Res 20 569–574 Occurrence Handle10.1080/00288330.1986.9516177

    Article  Google Scholar 

  • J. Thompson (1952) ArticleTitleThe acclimatization and growth of the Pacific oyster Crassostrea gigas in Australia Aust J Mar Freshwater Res 3 64–73

    Google Scholar 

  • J. Thompson (1959) ArticleTitleThe naturalization of the Pacific oyster in Australia Aust J Mar Freshwater Res 10 144–149

    Google Scholar 

  • J.D. Thompson T.J. Gibson F. Plewniak F. Jeanmougin D.G. Higgins (1997) ArticleTitleThe CLUSTAL X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res 25 4876–4882 Occurrence Handle1:CAS:528:DyaK1cXntFyntQ%3D%3D Occurrence Handle9396791

    CAS  PubMed  Google Scholar 

  • R.D. Ward L.J. English D.J. McGoldrick G.B. Maguire J.A. Nell P.A. Thompson (2000) ArticleTitleGenetic improvement of the Pacific oyster, Crassostrea gigas, in Australia Aquaculture Res 31 35–44 Occurrence Handle10.1046/j.1365-2109.2000.00388.x

    Article  Google Scholar 

  • J. Wuyts P. Rijk ParticleDe Y. Peer ParticleVan de T. Winkelmans R. Wachter ParticleDe (2001) ArticleTitleThe European Large Subunit Ribosomal RNA database Nucleic Acids Res 29 175–177 Occurrence Handle10.1093/nar/29.1.175 Occurrence Handle1:CAS:528:DC%2BD3MXjtlWmsro%3D Occurrence Handle11125083

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Dr. C.L. Hewitt, who was responsible for initiating the project. Drs. S. Appleyard, R. Ward, X. Guo, P. Boudry, and K. Reece provided DNA or tissue samples of many animals used in the study. Cultured larvae were supplied by Geordy River Aquaculture (St. Helens, Tasmania), and adults of the native flat oyster O. angasi came from Shellfish Culture Limited (Pipe Clay Lagoon, Tasmania). This project is part of the Hastings National Demonstration Project, jointly funded by the Commonwealth Natural Heritage Trust and Victorian Government. Partners in the project are Australian Quarantine and Inspection Service and CSIRO Marine Research. The shipping and port industries provided significant contributions to the project. The Environment Protection Authority (EPA) Victoria managed the project on behalf of the Victorian Government.

Author information

Authors and Affiliations

  1. CSIRO Marine Research, P.O. Box 1538, Hobart, Tasmania 7001, Australia

    Jawahar G. Patil, Rasanthi M. Gunasekera & Nicholas J. Bax

  2. School of Zoology, University of Tasmania, Private Bag 5, Tasmania 7001, Australia

    Bruce E. Deagle

Authors
  1. Jawahar G. Patil
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rasanthi M. Gunasekera
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bruce E. Deagle
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nicholas J. Bax
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jawahar G. Patil.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Patil, J.G., Gunasekera, R.M., Deagle, B.E. et al. Specific Detection of Pacific Oyster (Crassostrea gigas) Larvae in Plankton Samples Using Nested Polymerase Chain Reaction. Mar Biotechnol 7, 11–20 (2005). https://doi.org/10.1007/s10126-004-0034-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10126-004-0034-z

Keywords

Search

Navigation