Hostname: page-component-cd9895bd7-jkksz Total loading time: 0 Render date: 2024-12-26T21:22:12.090Z Has data issue: false hasContentIssue false
  • English
  • Français

Identification and quantification of two species of oyster larvae using real-time PCR⋆

Published online by Cambridge University Press:  15 January 2015

Ana Sánchez ,
Javier Quinteiro ,
Manuel Rey-Méndez ,
Ricardo Isaac Perez-Martín  and
Carmen González Sotelo
Ana Sánchez*
Affiliation:
Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas (CSIC), Eduardo Cabello 6, Vigo 36208 Pontevedra, Spain
Javier Quinteiro
Affiliation:
Laboratorio de Sistemática Molecular (Unidad Asociada al CSIC), Departamento de Bioquímica y Biología Molecular, CIBUS, Campus Vida, Universidad de Santiago de Compostela, Santiago de Compostela 15782 A Coruña, Spain
Manuel Rey-Méndez
Affiliation:
Laboratorio de Sistemática Molecular (Unidad Asociada al CSIC), Departamento de Bioquímica y Biología Molecular, CIBUS, Campus Vida, Universidad de Santiago de Compostela, Santiago de Compostela 15782 A Coruña, Spain
Ricardo Isaac Perez-Martín
Affiliation:
Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas (CSIC), Eduardo Cabello 6, Vigo 36208 Pontevedra, Spain
Carmen González Sotelo
Affiliation:
Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas (CSIC), Eduardo Cabello 6, Vigo 36208 Pontevedra, Spain
*
a Corresponding author: [email protected]
Get access

Abstract

A real-time polymerase chain reaction (PCR) assay was developed for the identification and quantification of two oyster species: Ostrea edulis and Crassostrea gigas. Two sets of primers and TaqMan-MGB probes were designed, based on partial sequences of the 16S rRNA gene. An amplification positive control system was also located in the 18S rRNA gene sequences. Closely related species of oysters and other bivalves, known to co-occur with the target species in European waters, were used to test the assay for cross-reactivity. The assay designed was specific for the target species and no signal or no significant signal was detected for all non-target species tested. The high sensitivity of this method was demonstrated since it is possible to detect just one larva (150–200 μm size) of each species even when it is present with others. Furthermore, this assay provided an acceptable quantification of the number of spiked larvae (1, 10 and 100 larvae) in plankton samples employing a standard curve for larvae.

Keywords

Real-time PCROyster larvae identificationSpecies identification16S rRNA
Type
Research Article
Information
Aquatic Living Resources , Volume 27 , Issue 3-4 , July 2014 , pp. 135 - 145
Copyright
© EDP Sciences, IFREMER, IRD 2015

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

⋆

Supporting information is only available in electronic form at www.alr-journal.org.

References

André, C.,Lindegarth, M.,Jonsson, P.R.,Sundberg, P., 1999, Species identification of bivalve larvae using random amplified polymorphic (RAPD): differentiation between Cerastoderma edule and C. lamarcki. J. Mar. Biol. Assoc. UK 79, 563–565. CrossRefGoogle Scholar
Applied Biosystems, 2005, Real-Time PCR Systems. Chemistry Guide.
Bell, J.L.,Grassle, J.P., 1998, A DNA probe for identification of larvae of the commercial surfclam (Spisula solidissima). Mol. Mar. Biol. Biotechnol. 7, 127–137. Google Scholar
Bendezu, I.F.,Slater, J.W.,Carney, B.F., 2005, Identification of Mytilus spp. and Pecten maximus in Irish waters by standar PCR of the 18S rDNA gene and multiplex PCR of the 16S rDNA gene. Mar. Biotechnol. 7, 687–696. CrossRefGoogle Scholar
Dias, P.J.,Sollelis, L.,Cook, E.J.,Piertney, S.B., Davies, I.M.,Snow,, M., 2008, Development a real-time PCR assay for detection of Mytilus species specific alleles: Application to a sampling survey in Scotland. J. Exp. Mar. Biol. Ecol. 367, 253–258. CrossRefGoogle Scholar
Figueiras, F.G.,Labarta, U.,Fernandez-Reiriz, M.J., 2002, Coastal upwelling, primary production and mussel growth in the Rias Baixas of Galicia. Hydrobiologia 484, 121–131. CrossRefGoogle Scholar
Garland, E.D.,Zimmer, C.A., 2002, Techniques for the identification of bivalve larvae. Mar. Ecol. Prog. Ser. 225, 299–310. CrossRefGoogle Scholar
Goodwin, J.D.,North, E.W.,Thompson, C.M., 2014, Evaluating and improving a semi-automated image analysis technique for identifying bivalve larvae. Limnol. Oceanogr. Methods 12, 548–562. CrossRefGoogle Scholar
Hall, T.A., 1999, BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98. Google Scholar
Hare, M.P.,Palumbi, S.R.,Butman, C.A., 2000, Single-step species identification of bivalve larvae using multiplex polymerase chain reaction. Mar. Biol. 137, 953–961. CrossRefGoogle Scholar
Henzler, C.M.,Hoaglund, E.A.,Gaines, S.D., 2010, FISH-CS- A rapid method for counting and sorting species of marine zooplankton. Mar. Ecol. Prog. Ser. 410, 1–11. CrossRefGoogle Scholar
Hosoi, M.,Hosoi-Tanabe, S.,Sawada, H.,Ueno, M.,Toyohara, H.,Hayashi, I., 2004, Sequence and polymerase chain reaction-restriction fragment length polymorphism analysis of the large subunit rRNA gene of bivalve: Simple and widely applicable technique for multiple species identification of bivalve larva. Fish. Sci. 70, 629–637. CrossRefGoogle Scholar
Hosoi-Tanabe, S.,Sako, Y., 2005, Species-specific detection and quantification of toxic marine dinoflagellates Alexandrium tamarense and A. catenella by real-time PCR assay. Mar. Biotechnol. 7, 506–514. CrossRefGoogle Scholar
Hurwood, D.A.,Heasman, M.P.,Mather, P.B., 2005, Gene flow, colonisation and demographic history of the flat oyster Ostrea angasi. Mar. Freshw. Res. 56, 1099–1106. CrossRefGoogle Scholar
Iglesias, D.,Rodríguez, L.,Montes, J.,Conchas, R.F.,Pérez, J.L.,Fernández, M.,Guerra, A., 2005, Estudio de viabilidad del cultivo de ostra rizada Crassostrea gigas (Thunberg, 1793) en diferentes rías gallegas. Primeros resultados biológico-productivos. Bol. Inst. Esp. Oceanogr. 21, 293–309. Google Scholar
Johnson, M.,Zaretskaya, I.,Raytselis, Y.,Merezhuk, Y.,McGinnis, S.,Madden, T.L., 2008, NCBI BLAST: a better web interface. Nucleic Acid Res. 36 (Suppl. 2), W5-W9. CrossRefGoogle Scholar
Jozefowicz, C.J., ÓFoighil, D., 1998, Phylogenetic analysis of southern hemisphere flat oysters based on partial mitochondrial 16S rDNA gene sequences. Mol. Phyl. Evol. 10, 426–435. CrossRefGoogle ScholarPubMed
Kenchington, E.,Bird, C.J.,Osborne, J.,Reith, M., 2002, Novel repeat elements in the nuclear ribosomal RNA operon of the flat oysters O. edulis C. Linnaeus, 1758 and O. angasi Sowerby, 1871. J. Shellfish Res. 21, 697–705. Google Scholar
Le Goff-Vitry, M.C.,Chipman, A.R.,Comtet, T., 2007, In situ hybridization on whole larvae: a novel method for monitoring bivalve larvae. Mar. Ecol. Prog. Ser. 343, 161–172. CrossRefGoogle Scholar
Lorenzo-Abalde, S.,González-Fernández, A., De Miguel Villegas, E.,Fuentes, J., 2005, Two monoclonal antibodies for the recognition of Mytilus spp. larvae: Studies on cultured larvae and tests on plankton samples. Aquaculture 250, 736–747. CrossRefGoogle Scholar
Mirella da Silva, P.,Fuentes, J.,Villalba, A., 2005, Growth, mortality and disease susceptibility of oyster Ostrea edulis families obtained from brood stocks of different geographical origins, through on-growing in the Ria de Arousa (Galicia, NW Spain). Mar. Biol. 147, 965–977. CrossRefGoogle Scholar
Palumbi S.R., Martin A., Romano S., McMillan W.O., Stice L., Grabowski G., 1991, The simple Fool’s Guide to PCR. Department of Zoology, University of Hawaii, Honolulu.
Pan, M.,McBeath, A.J.A.,Hay, S.J.,Pierce, G.J.,Cunningham, C.O., 2008, Real-time PCR assay for detection and relative quantification of Liocarcinus depurator larvae from plankton samples. Mar. Biol. 153, 859–870. CrossRefGoogle Scholar
Patil, J.G.,Gunasekera, R.M.,Deagle, B.E.,Bax, N.J., 2005, Specific detection of Pacific oyster (Crassostrea gigas) larvae in plankton samples using nested polymerase chain reaction. Mar. Biotech. 7, 11–20. CrossRefGoogle ScholarPubMed
Paugam, A., Le Pennec, M.,Geneviéve, A.F., 2000, Immunological recognition of marine bivalve larvae from plankton samples. J. Shellfish Res. 19, 325–331. Google Scholar
Paugam, A., Le Pennec, M.,Marhic, A.,Geneviéve, A.F., 2003, Immunological in situ determination of Pecten maximus larvae and their temporal distribution. J. Mar. Biol. Assoc. UK 83, 1083–1093. CrossRefGoogle Scholar
Pérez, D.,Lorenzo-Abalde, S.,González-Fernández, A.,Fuentes, J., 2009, Immunodetection of Mytilus galloprovincialis larvae using monoclonal antibodies to monitor larval abundance on the Galician coast: Optimization of the method and comparison with identification by morphological traits. Aquaculture 294, 86–92. CrossRefGoogle Scholar
Quinteiro, J.,Pérez-Diéguez, L.,Sánchez, A., Pérez-Martín, R.I.,Sotelo, C.G,Rey-Méndez, M., 2011, Quantification of manila clam Ruditapes philippinarum (Adams & Reeve, 1850) larvae based on SYBR Green real-time polymerase chain reaction. J. Shellfish Res. 30, 791–796. CrossRefGoogle Scholar
Reece, K.S.,Cordes, J.F.,Stubbs, J.B.,Hudson, K.L.,Francis, E.A., 2008, Molecular phylogenies help resolve taxonomic confusion with Asian Crassostrea oyster species. Mar. Biol. 153, 709–721. CrossRefGoogle Scholar
Smith, K.F.,Wood, S.A.,Mountfort, D.O.,Cary, S.C., 2012, Development of a real-time PCR assay for the detection of the invasive clam, Corbula amurensis, in environmental samples. J. Exp. Mar. Biol. Ecol. 412, 52–57. CrossRefGoogle Scholar
Thompson, J.D.,Gibson, T.J.,Plewniak, F.,Jeanmougin, F.,Higgins, D.G., 1997, The CLUSTAL X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids. Res. 25, 4876–4882. CrossRefGoogle ScholarPubMed
Thompson, C.M.,Hare, M.P.,Gallager, S.M., 2012, Semi-automated image analysis for the identification of bivalve larvae from a Cape Cod estuary. Limnol. Oceanogr. Methods 10, 538–554. CrossRefGoogle Scholar
Toro, J.E., 1998, Molecular identification of four species of mussels from southern Chile by PCR-based nuclear markers: The potential use in studies involving planktonic surveys. J. Shellfish Res. 17, 1203–1205. Google Scholar
Vadopalas, B.,Bouma, J.V.,Jackels, C.R.,Friedman, C.S., 2006, Application of real-time PCR for simultaneous identification and quantification of larval abalone. J. Exp. Mar. Biol. Ecol. 334, 219–228. CrossRefGoogle Scholar
Wang, S.,Bao, Z.,Zhang, L.,Li, N.,Zhan, A.,Guo, W.,Wang, X.,Hu, J., 2006, A new strategy for species identification of planktonic larvae: PCR-RFLP analysis of the internal transcribed spacer region of ribosomal DNA detected by agarose gel electrophoresis or DHPLC. J. Plankton Res. 28, 375–384. CrossRefGoogle Scholar
Wight, N.A.,Suzuki, J.,Vadopalas, B.,Friedman, C.S., 2009, Development and optimization of quantitative PCR assays to aid Ostrea lurida carpenter 1864 restoration efforts. J. Shellfish Res. 28, 33–41. CrossRefGoogle Scholar
Yuan J.S., Reed A., Chen F., Stewart Jr. C.N., 2006, Statistical analysis of real-time PCR data. BMC Bioinformatics 7.