Skip to main content
SpringerLink
Log in

Real-time PCR assay for detection and relative quantification of Liocarcinus depurator larvae from plankton samples

  • Research Article
  • Published:
Marine Biology Aims and scope Submit manuscript

Abstract

Accurate species identification of decapod crustacean larvae is required to understand their population distributions, life cycle dynamics and interactions with their habitats. Analysis of plankton samples using morphological taxonomic methods and microscopy is time-consuming, requires highly skilled and trained operatives and may often be inaccurate. As complementary tools to classical identification methods, recent work has focused on the development of molecular approaches and shows their feasibility for species-specific identification. This study has developed real-time PCR assays utilising species-specific Taqman® probes designed in the cytochrome oxidase I (COI) gene of Liocarcinus depurator, Necora puber, Carcinus maenas and Cancer pagurus. Our study then employed the probe and primers designed for L. depurator to obtain accurate identification and relative abundance estimates of L. depurator larvae in plankton samples collected between March 2005 and October 2006. Ranges of larval abundances were derived from a standard curve created from plankton samples spiked with a known number of larvae reared in the laboratory. Inhibition of the PCR reaction was shown to be an important factor and our results suggested that 0.1 ng of DNA as template provided accurate identification and avoided inhibition. Real-time PCR was shown to provide accurate species identification on unsorted plankton samples and could be suitable for the estimation of larval abundances in the plankton, although more work must be done to improve the accuracy of those estimations.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Anger K (2001) The biology of decapod crustacean larvae. crustacean issues, vol 14. A.A. Balkema, The Netherlands

    Google Scholar 

  • Bot PVM, van Raaphorst W, Batten S, Laane RWPM, Philippart K, Radach G, Frohse A, Schultz H, van den Eynde D, Colijn F (1996) Comparison of changes in the annual variability of the seasonal cycles of chlorophyll, nutrients and zooplankton at eight locations on the north-west european continental shelf. German J Hydrogr 48:349–364

    Google Scholar 

  • Bromley PJ, Watson T, Hislop JRG (1997) Diel feeding patterns and the development of food webs in pelagic 0-group cod (Gadus morhua L.), haddock (Melanogrammus aeglefinus, L.), whiting (Merlangius merlangus L.), saithe (Pollachius virens L.), and Norway pout (Trisopterus esmakii Nilson) in the northern North Sea. ICES J Mar Sci 54:846–853

    Article  Google Scholar 

  • Bucklin A, Guarnieri M, Hill R, Bentley A, Kaartvedt S (1999) Taxonomic and systematic assessment of planktonic copepods using mitochondrial COI sequence variation and competitive species-specific PCR. Hydrobiologia 401:239–254

    Article  CAS  Google Scholar 

  • Clark PF (1984) A comparative study of zoeal morphology in the genus Liocarcinus (Crustacea: Brachyura: Portunidae). Zool J Linn Soc-Lond 82:273–290

    Article  Google Scholar 

  • Deagle BE, Bax N, Hewitt CL, Patil JG (2003) Development and evaluation of a PCR-based test for detection of Asterias (Echinodermata: Asteroidea) larvae in Australian plankton samples from ballast water. Mar Freshw Res 54:709–719

    Article  CAS  Google Scholar 

  • d’Udekem d’Acoz C (1999) Inventaire et distribution des crustacés décapodes de l’Atlantique nord-oriental, de la Méditerranée et des eaux continentals adjacentes au nord de 25°N. Patrimoines naturels (M.N.H.N./S.P.N.), 40:383 p

  • Eimanifar A, Rezvani S, Carapetian J (2006) Application of RFLP analysis to identify cyst populations of Artemia urmiana Günther, 1899 (Branchiopoda, Anostraca) from Urmia Lake, Iran. Crustaceana 78:1311–1323

    Article  Google Scholar 

  • Ellison CK, Burton RS (2005) Application of bead array technology to community dynamics of marine phytoplankton. Mar Ecol Prog Ser 288:75–85

    Article  CAS  Google Scholar 

  • Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mithocondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299

    PubMed  CAS  Google Scholar 

  • Fox CJ, Taylor MI, Pereyra R, Villasana MI, Rico C (2005) TaqMan DNA technology confirms likely overestimation of cod (Gadus morhua L.) egg abundance in the Irish Sea: implications for the assessment of the cod stock and mapping of spawning areas using egg-based methods. Mol Ecol 14:879–884

    Article  PubMed  CAS  Google Scholar 

  • Gimenez L (2006) Phenotypic links in complex life cycles: conclusions from studies with decapod crustaceans. Integr Comp Biol 46:615–622

    Article  Google Scholar 

  • Hare MP, Palumbi SR, Butman CA (2000) Single-step species identification of bivalve larvae using multiplex polymerase chain reaction. Mar Biol 137:953–961

    Article  CAS  Google Scholar 

  • Hill RS, Allen LD, Bucklin A (2001) Multiplexed species-specific PCR protocol to discriminate four N. Atlantic Calanus species, with an mtCOI gene tree for ten Calanus species. Mar Biol 139:279–287

    Article  CAS  Google Scholar 

  • Hebert PDN, Ratnasingham S, deWaard JR (2003) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc R Soc Lond B (Suppl) 270:S96–S99

    Article  CAS  Google Scholar 

  • Hughes G, Beaumont AR (2004) A potential method for discriminating between tissue from the European Lobster (Homarus gammarus) and the American Lobster (H. americanus). Crustaceana 77:371–376

    Article  Google Scholar 

  • Ingle R (1992) Larval stages of northeastern Atlantic crabs. Chapman & Hall, Natural History Museum publications, London

    Google Scholar 

  • Kim KB, Hong SY (1999) Larval development of the wrinkled swimming crab Liocarcinus corrugatus (Decapoda: Brachyura: Portunidae) reared in the laboratory. J Crust Biol 19:792–808

    Article  Google Scholar 

  • Lindeque PK, Harris RP, Jones MB, Smerdon GR (1999) Simple molecular method to distinguish the identity of Calanus species (Copepoda: Calanoida) at any developmental stage. Mar Biol 133:91–96

    Article  CAS  Google Scholar 

  • Lindeque PK, Harris RP, Jones MB, Smerdon GR (2004) Distribution of Calanus spp. as determined using a genetic identification system. Sci Mar 68:121–128

    Article  Google Scholar 

  • Lindeque PK, Hay SJ, Heath MR, Ingvarsdottir A, Rasmussen J, Smerdon GR, Waniek JJ (2006) Integrating conventional microscopy and molecular analysis to analyse the abundance and distribution of four Calanus congeners in the North Atlantic. J Plank Res 28:221–238

    Article  CAS  Google Scholar 

  • Martin J (2000) Les larves de crustacés décapodes des côtes françaises de la Manche. Identification, période, abondance. Ifremer, Plouzané, France

  • McBeath AJA, Penston MJ, Snow M, Cook PF, Bricknell IR, Cunningham CO (2006) Development and application of real-time PCR for specific detection of Lepeophtheirus salmonis and Caligus elongatus larvae in Scottish plankton samples. Dis Aquat Organ 73:141–150

    Article  PubMed  CAS  Google Scholar 

  • Medeiros-Bergen DE, Olson RR, Conroy JA, Kocher TD (1995) Distribution of holothurian larvae determined with species-specific genetic probes. Limnol Oceanogr 40:1225–1235

    Article  Google Scholar 

  • Øines O, Heuch PA (2005) Identification of sea louse species of the genus Caligus using mtDNA. J Mar Biol Ass UK 85:73–79

    Article  CAS  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007

    Article  Google Scholar 

  • Raymont JEG (1963) Plankton and productivity in the oceans. 2nd edn., vol 2. Pergamon, Zooplankton

    Google Scholar 

  • Siokou-Frangou I (1996) Zooplankton annual cycle in a Mediterranean coastal area. J Plank Res 18:203–223

    Article  Google Scholar 

  • Taylor MI, Fox C, Rico I, Rico C (2002) Species-specific TaqMan probes for simultaneous identification of cod (Gadus morhua L.), haddock (Melanogrammus aeglefinus L.) and whiting (Merlangius merlangus L.). Mol Ecol Notes 2:599–601

    Article  CAS  Google Scholar 

  • Vadopalas B, Bouma JV, Jackels CR, Friedman CS (2006) Application of real-time PCR for simultaneous identification and quantification of larval abalone. J Exp Mar Biol Ecol 334:219–228

    Article  CAS  Google Scholar 

  • Valdes L, Moral M (1998) Time-series analysis of copepod diversity and species richness in the southern Bay of Biscay off Santander, Spain, in relation to environmental conditions. ICES J Mar Sci 55:783–792

    Article  Google Scholar 

  • van Guelpen L, Markle DF, Duggan DJ (1982) An evaluation of accuracy, precision, and speed of several zooplankton subsampling techniques. ICES J Mar Sci 40:226–236

    Article  Google 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 Plank Res 28:375–384

    Article  Google Scholar 

  • Watanabe S, Minegishi Y, Yoshinaga T, Aoyama J, Tsukamoto K (2004) A quick method for species identification of Japanese Eel (Anguilla japonica) using Real-Time PCR: an onboard application for use during sampling surveys. Mar Biotechnol 6:566–574

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank J. Dunn and Temora crew (R. Cargill, M. Leys, A. MacDonald, P. MacDonald, T. Morrice, J. Smith) for their efforts and invaluable help in collecting plankton samples and supplying ovigerous females.

Author information

Authors and Affiliations

  1. FRS Marine Laboratory, PO Box 101, Victoria Rd, Aberdeen, AB11 9DB, UK

    Maria Pan, Alastair J. A. McBeath, Steve J. Hay & Carey O. Cunningham

  2. Oceanlab, University of Aberdeen, Main Street, Newburgh, Aberdeenshire, AB41 6AA, UK

    Graham J. Pierce

Authors
  1. Maria Pan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alastair J. A. McBeath
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Steve J. Hay
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Graham J. Pierce
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Carey O. Cunningham
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Maria Pan.

Additional information

Communicated by A. Atkinson.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pan, M., McBeath, A.J.A., Hay, S.J. et al. Real-time PCR assay for detection and relative quantification of Liocarcinus depurator larvae from plankton samples. Mar Biol 153, 859–870 (2008). https://doi.org/10.1007/s00227-007-0858-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00227-007-0858-y

Keywords

Search

Navigation