Skip to main content
SpringerLink
Log in

Measurement of copepod predation on nauplii using qPCR of the cytochrome oxidase I gene

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

Abstract

A method to directly measure predation rates by older stage copepods upon copepod nauplii using species-specific primers for the mitochondrial cytochrome oxidase subunit one gene (mtCOI) and real-time quantitative PCR (qPCR) was developed. The general approach is to determine the mtCOI gene copy number of an individual prey organism and the copy number of the same gene in the stomachs of predatory copepods collected in the field. From the knowledge of DNA disappearance rates in the stomachs, ingestion rates can be calculated. In October 2006, laboratory experiments were carried out with Acartia tonsa N1 and N2 as prey and adult female Centropages typicus as predator. The copepods were collected in Narragansett Bay, USA. A. tonsa mtCOI copy numbers copepod−1 were determined for stages N1–C1 and for adults. A. tonsa DNA was detectable in the guts of the predators for as long as 3 h. Exponential rates of decline in prey DNA from the stomachs of the predators are similar to those measured for gut pigments. Because of the very small amount of DNA in an individual N1 or N2 nauplius, procedures were developed to maximize the quantitative extraction and recovery of DNA and to increase the sensitivity of the method. Two quite divergent haplotypes of A. tonsa were found in Narragansett Bay, which required separate qPCR primers; one was present in summer (July) and the other in fall and winter (October and February). With modification, the methods in this study can likely be applied to a range of predator–prey systems.

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

  • Berggreen U, Hansen B, Kiorboe T (1988) Food size spectra, ingestion and growth of the copepod Acartia tonsa during development: implications for determination of copepod production. Mar Biol 99:341–352

    Article  Google Scholar 

  • Besiktepe S, Dam HJ (2002) Coupling of ingestion and defecation as a function of diet in the calanoid copepod Acartia tonsa. Mar Ecol Prog Ser 229:151–164

    Article  Google Scholar 

  • Blankenship LE, Yayanos AA (2005) Universal primers and PCR gut contents to study marine invertebrate diets. Mol Ecol 14:891–899

    Article  CAS  Google Scholar 

  • Boleyn BJ (1967) A simple pipette control for isolation planktonic algae. Can J Microbiol 13:1129–1130

    Article  CAS  Google Scholar 

  • Bowers HA, Tengs T, Glasgow HB Jr, Burkholder JM, Rublee PA, Oldach DW (2000) Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates. Appl Environ Microbiol 66:4641–4648

    Article  CAS  Google Scholar 

  • Caudill CC, Bucklin A (2004) Molecular phylogeography and evolutionary history of the estuarine copepod, Acartia tonsa, on the Northwest Atlantic coast. Hydrobiologia 511:91–102

    Article  Google Scholar 

  • Coyne KJ, Handy SM, Demir E, Whereat EB, Hutchins DA, Portune KJ, Doblin MA, Cary SC (2005) Improved quantitative real-time PCR assays for enumeration of harmful algal species in field samples using an exogenous DNA reference standard. Limnol Oceanogr Methods 3:381–391

    Article  CAS  Google Scholar 

  • Dam HG, Peterson WT (1988) The effect of temperature on the gut clearance rate constant of planktonic copepods. J Exp Mar Biol Ecol 123:1–14

    Article  Google Scholar 

  • Durbin EG, MC Casas (2006) Abundance and spatial distribution of copepods on Georges Bank during the winter/spring period. Deep Sea Res Part II 53:2537–2569

    Article  Google Scholar 

  • Durbin EG, Campbell RG (2007) Reassessment of the gut pigment method for estimating in situ zooplankton ingestion. Mar Ecol Progr Ser 331:305–307

    Google Scholar 

  • Durbin AG, Durbin EG, Wlodarczyk E (1990) Diel feeding behavior in the marine copepod Acartia tonsa in relation to food avaliability. Mar Ecol Progr Ser 68:23–45

    Article  Google Scholar 

  • Durbin EG, Campbell RW, Gilman S, Durbin AG (1995) In situ feeding of Calanus finmarchicus in the southern Gulf of Maine during late spring. Cont Shelf Res 15:539–570

    Article  Google Scholar 

  • Dyhrman ST, Erdner D, La Du J, Galac M, Anderson DM (2006) Molecular quantification of toxic Alexandrium fundyense in the Gulf of Maine using real-time PCR. Harmful Algae 5:242–250

    Article  CAS  Google Scholar 

  • Eiane K, Ohman MD (2004) Stage-specific mortality of Calanus finmarchicus, Pseudocalanus elongatus and Oithona similis on Fladen Ground, North Sea, during a spring bloom. Mar Ecol Progr Ser 268:183–193

    Article  Google Scholar 

  • Elliott JM, Persson L (1978) The estimation of daily rates of food consumption for fish. J Anim Ecol 47:977–991

    Article  Google Scholar 

  • Ellis SG, Small LF (1989) Comparison of gut evacuation rates of feeding and non-feeding Calanus marshallae. Mar Biol 103:175–181

    Article  Google Scholar 

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

    CAS  Google Scholar 

  • Galluzzi L, Penna A, Bertozzini E, Vila M, Garces E, Magnani M (2004) Development of real-time PCR assay for rapid detection and quantification of Alexandrium minutum (a dinoflagellate). Appl Environ Microbiol 70:1199–1206

    Article  CAS  Google Scholar 

  • Gray M, Wawrik B, Paul J, Casper E (2003) Molecular detection and quantification of the red tide dinoflagellate Karenia brevis in the marine environment. Appl Environ Micriobiol 69:5726–5730

    Article  CAS  Google Scholar 

  • Handy SM, Hutchins DA, Cary SC, Coyne KJ (2006) Simultaneous enumeration of multiple raphidophyte species by quantitative real-time PCR: capabilities and limitations. Limnol Oceanogr: Methods 4:193–204

    Article  Google Scholar 

  • Head EJH, Harris LR (1994) Feeding selectivity of copepods grazing on natural mixtures of phytoplankton by HPLC analysis of pigments. Mar Ecol Progr Ser 110:75–83

    Article  CAS  Google Scholar 

  • Hebert PDN, Cywinska A, Ball SL, JdeWaard JR (2002) Biological identifications through DNA barcodes. Proc Roy Soc B 02PB0653.5

  • Irigoien X (1998) Gut clearance rate constant, temperature and initial gut contents: a review. J Plankton Res 20:997–1003

    Article  Google Scholar 

  • Irigoien X, Harris RP (2006) Comparative population structure, abundance and vertical distribution of six copepod species in the North Atlantic: evidence for intraguild predation? Mar Biol Res 2:276–290

    Article  Google Scholar 

  • Jarman SN, Gales NJ, Tierney M, Gill PC, Elliott NG (2002) A DNA-based method for identification of krill species and its application to analyzing the diet of marine vertebrate predators. Mol Ecol 11:2679–2690

    Article  CAS  Google Scholar 

  • Juhl AR, Ohman MD, Goericke R (1996) Astaxanthin in Calanus pacificus: assessment of pigment-based measures of omnivory. Limnol Oceanogr 41:1198–1207

    Article  CAS  Google Scholar 

  • Kleppel GS, Pieper RE (1984) Phytoplankton pigments in the gut contents of planktonic copepods from coastal waters off southern California. Mar Biol 78:193–198

    Article  CAS  Google Scholar 

  • Kleppel GS, Frazel D, Pieper RE, Holliday DV (1988) Natural diets of zooplankton off southern California. Mar Ecol Progr Ser 49:231–241

    Article  Google Scholar 

  • Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–20

    Article  CAS  Google Scholar 

  • Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–51

    Article  CAS  Google Scholar 

  • Mackas DL, Bohrer R (1976) Fluorescence analysis of zooplankton gut contents and an investigation of diel feeding patterns. J Exp Mar Biol Ecol 25:77–85

    Article  Google Scholar 

  • Mauchline J (1998) The biology of plankton copepods. Adv mar biol vol 33 Academic, San Diego

    Google Scholar 

  • Nejstgaard JC, Frischer ME, Raule CL, Grubel R, Kohlberg KE, Verity PG (2003) Molecular detection of algal prey in copepod guts and fecal pellets. Limnol Oceanogr Methods 1:29–38

    Article  Google Scholar 

  • Ohman MD (1992) Immunochemical recognition of oligotrich ciliates. Mar Biol 114:653–660

    Article  Google Scholar 

  • Ohman MD, Hirche HJ (2001) Density-dependent mortality in an oceanic copepod population. Nature 412:638–641

    Article  CAS  Google Scholar 

  • Ohman MD, Wood SN (1995) The inevitability of mortality. ICES J Mar Sci 52:517–522

    Article  Google Scholar 

  • Ohman MD, Runge JA, Durbin EG Field DB, Niehoff B (2002) On birth and death in the sea. Hydrobiologia 480:55–68

    Article  Google Scholar 

  • Ohman MD, Sullivan BK, Durbin EG, Runge JA (2005) Relationship of predation potential to mortality for Calanus finmarchicus on Georges Bank, N.W. Atlantic. ASLO Summer Meeting, June 2005

  • Olson RJ, Mullen AJ (1986) Recent developments for making gastric evacuation and daily ration determinations. Environ Biol Fishes 16:183–191

    Article  Google Scholar 

  • Popels LC, Cary SC, Hutchins DA, Forbes R, Pustizzi F, Gobler CJ, Coyne KJ (2003) The use of quantitative polymerase chain reaction for the detection and enumeration of the harmful alga Aureococcus anophagefferens in environmental samples along the United States East Coast. Limnol Oceanogr Methods 1:92–102

    Article  Google Scholar 

  • Read DS, Sheppard SK, Bruford MW, Glen DM, Symondson WOC (2006) Molecular detection of predation by soil micro-arthropods on nematodes. Mol Ecol 15:1963–1972

    Article  CAS  Google Scholar 

  • Saito K, Drgon K, Robledo J, Krupatkina D, Vasta G (2002) Characterization of the rRNA locus of Pfiesteria piscicida and development of standard and quantitative PCR-based detection. Appl Environ Microbiol 68:5394–5407

    Article  CAS  Google Scholar 

  • Sell AF, van Keuren D, Madin LP (2001) Predation by omnivorous copepods on early developmental stages of Calanus finmarchicus and Pseudocalanus spp. Limnol Oceanogr 46:953–959

    Article  Google Scholar 

  • Symondson WOC (2002) Molecular identification of prey in predator diets. Mol Ecol 11:627–641

    Article  CAS  Google Scholar 

  • Tirelli V, Mayzaud P (2005) Relationship between functional response and gut transit time in the calanoid copepod A. clausi: role of food quality and quantity. J Plankton Res 27:557–568

    Article  Google Scholar 

  • Titelman J, Fiksen O (2004) Ontogenetic vertical distribution patterns in small copepods: field observations and model predictions. Mar Ecol Prog Ser 284:49–63

    Article  Google Scholar 

  • Troedsson C, Frischer ME, Nejstgaard JC, Thompson EM (2007) Molecular quantification of differential ingestion and particle trapping rates by the appendicularian Oikopleura dioica as a function of prey size and shape. Limnol Oceanogr 52:416–427

    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 Mar Exp Biol Ecol 244:219–228

    Article  Google Scholar 

  • Vestheim H, Edvardsen B, Kaardvedt S (2005) Assessing feeding of a carnivorous copepod using species-specific PCR. Mar Biol 147:381–385

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We wish to thank the URI GSC Center for making their facilities available to us. This research was supported by NSF and NOAA.

Author information

Authors and Affiliations

  1. Graduate School of Oceanography, URI, South Ferry Rd, Narragansett, RI, 02882, USA

    Edward G. Durbin, Maria C. Casas, Tatiana A. Rynearson & David C. Smith

Authors
  1. Edward G. Durbin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria C. Casas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tatiana A. Rynearson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David C. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Edward G. Durbin.

Additional information

Communicated by J.P. Grassle.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Durbin, E.G., Casas, M.C., Rynearson, T.A. et al. Measurement of copepod predation on nauplii using qPCR of the cytochrome oxidase I gene. Mar Biol 153, 699–707 (2008). https://doi.org/10.1007/s00227-007-0843-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00227-007-0843-5

Keywords

Search

Navigation