Marine Biology

, Volume 156, Issue 3, pp 253–259 | Cite as

Quantification of copepod gut content by differential length amplification quantitative PCR (dla-qPCR)

  • Christofer TroedssonEmail author
  • Paolo Simonelli
  • Verena Nägele
  • Jens C. Nejstgaard
  • Marc E. Frischer
Original Paper


Quantification of feeding rates and selectivity of zooplankton is vital for understanding the mechanisms structuring marine ecosystems. However, methodological limitations have made many of these studies difficult. Recently, molecular based methods have demonstrated that DNA from prey species can be used to identify zooplankton gut contents, and further, quantitative gut content estimates by quantitative PCR (qPCR) assays targeted to the 18S rRNA gene have been used to estimate feeding rates in appendicularians and copepods. However, while standard single primer based qPCR assays were quantitative for the filter feeding appendicularian Oikopleura dioica, feeding rates were consistently underestimated in the copepod Calanus finmarchicus. In this study, we test the hypothesis that prey DNA is rapidly digested after ingestion by copepods and describe a qPCR-based assay, differential length amplification qPCR (dla-qPCR), to account for DNA digestion. The assay utilizes multiple primer sets that amplify different sized fragments of the prey 18S rRNA gene and, based on the differential amplification of these fragments, the degree of digestion is estimated and corrected for. Application of this approach to C. finmarchicus fed Rhodomonas marina significantly improved quantitative feeding estimates compared to standard qPCR. The development of dla-qPCR represents a significant advancement towards a quantitative method for assessing in situ copepod feeding rates without involving cultivation-based manipulation.


Calanoid Copepod Digestion Profile Standard qPCR High Pure Plasmid Isolation Target Gene Copy Number 



This work was supported by the Norwegian Research Council (NRC) project 145326/432 to C. T., NRC project 152714/120 30 to J. C. N. and the US National Science Foundation grants OPP-00-83381, OCE-08-25999 and the US Department of Energy Biotechnology Investigations—Ocean Margins Program (FG02-98EF 62531) to M. E. F. Special thanks to Tina Walters for invaluable technical assistance and two anonymous reviewers and Bruce Deagle for valuable comments that have significantly improved this manuscript. Anna Boyette prepared the figures.

Open Access

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


  1. Båmstedt U, Gifford DJ, Irigoien X, Atkinson A, Roman M (2000) Feeding. In: Harris R, Wiebe P, Lenz J, Skjoldal HR, Huntley M (eds) ICES zooplankton methodology manual. Academic Press, London, pp 297–399CrossRefGoogle Scholar
  2. Båmstedt U, Nejstgaard JC, Solberg PT (1999) Utilisation of small-sized food algae by Calanus finmarchicus (Copepoda, Calanoida) and the significance of feeding history. Sarsia 84:19–38CrossRefGoogle Scholar
  3. Blankenship LE, Yayanos AA (2005) Universal primers and PCR of gut contents to study marine invertebrate diets. Mol Ecol 14:891–899. doi: CrossRefGoogle Scholar
  4. Deagle BE, Eveson JP, Jarman SN (2006) Quantification of damage in DNA recovered from highly degraded samples—a case study on DNA in faeces. Front Zool 3:11. doi: CrossRefGoogle Scholar
  5. Durbin EG, Casas MC, Rynearson TA, Smith DC (2008) Measurement of copepod predation on nauplii using qPCR of the cytochrome oxidase I gene. Mar Biol (Berl) 153:699–707. doi: CrossRefGoogle Scholar
  6. Galluzzi L, Penna A, Bertozzini E, Giacobbe MG, Vila M, Garces E et al (2005) Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis). Harmful Algae 4:965–1130. doi: CrossRefGoogle Scholar
  7. Gruebl T, Frischer ME, Sheppard M, Neumann M, Maurer AN, Lee RF (2002) Development of an 18S rRNA gene targeted PCR based diagnostic for the blue crab parasite Hematodinium sp. Dis Aquat Organ 49:61–70. doi: CrossRefGoogle Scholar
  8. Guillard RRL (1975) Culture of phytoplankton for feeding marine invertebrates. In: Smith WL, Chanley MH (eds) Culture of marine invertebrate animals. Plenum Press, New York, pp 29–60CrossRefGoogle Scholar
  9. Harper GL, King RA, Dodd CS, Harwood JD, Glen DM, Bruford MW et al (2005) Rapid screening of invertebrate predators for multiple prey DNA targets. Mol Ecol 14:819–827. doi: CrossRefGoogle Scholar
  10. Harwood JD, Obrycki JJ (2005) Quantifying aphid predation rates of generalist predators in the field. Eur J Entomol 102:335–350CrossRefGoogle Scholar
  11. Hoogendoorn M, Heimpel GE (2001) PCR-based gut content analysis of insect predators: using ribosomal ITS-1 fragments from prey to estimate predation frequency. Mol Ecol 10:2059–2067. doi: CrossRefGoogle Scholar
  12. Jarman SN, Deagle BE, Gales NJ (2004) Group-specific polymerase chain reaction for DNA-based analysis of species diversity and identity in dietary samples. Mol Ecol 13:1313–1322. doi: CrossRefGoogle Scholar
  13. Jarman SN, Gales NJ, Tierney M, Gill PC, Elliott NG (2002) A DNA-based method for identification of krill species and its application to analysing the diet of marine vertebrate predators. Mol Ecol 11:2679–2690. doi: CrossRefGoogle Scholar
  14. Martin DL, Ross RM, Quetin LB, Murray AE (2006) Molecular approach (PCR-DGGE) to diet analysis in young Antarctic krill Euphausia superba. Mar Ecol Prog Ser 319:155–165. doi: CrossRefGoogle Scholar
  15. Nejstgaard JC, Båmstedt U, Bagøien E, Solberg PT (1995) Algal constraints on copepod grazing. Growth state, toxicity, cell size, and season as regulating factors. ICES J Mar Sci 52:347–357. doi: CrossRefGoogle Scholar
  16. Nejstgaard JC, Frischer ME, Raule CL, Gruebel R, Kohlberg KE, Verity PG (2003) Molecular detection of algal prey in copepod guts and faecal pellets. Limnol Oceanogr Methods 1:29–38CrossRefGoogle Scholar
  17. Nejstgaard JC, Frischer ME, Simonelli P, Troedsson C, Brakel M, Adiyaman F et al (2008) Quantitative PCR to estimate copepod feeding. Mar Biol (Berl) 153:565–577. doi: CrossRefGoogle Scholar
  18. Sheppard SK, Harwood JD (2005) Advances in molecular predator–prey ecology. Funct Ecol 19:751–762. doi: CrossRefGoogle Scholar
  19. Symondson WOC (2002) Molecular identification of prey in predator diets. Mol Ecol 11:627–641. doi: CrossRefGoogle Scholar
  20. 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 52:416–427CrossRefGoogle Scholar
  21. Verity PG, Smetacek V (1996) Organism life cycles, predation, and the structure of marine pelagic ecosystems. Mar Ecol Prog Ser 130:277–293. doi: CrossRefGoogle Scholar
  22. Vestheim H, Edvardsen B, Kaartvedt S (2005) Assessing feeding of a carnivorous copepod using species specific PCR. Mar Biol (Berl) 147:381–385. doi: CrossRefGoogle Scholar
  23. Vestheim H, Jarman SN (2008) Blocking primers to enhance PCR amplification of rare sequences in mixed samples–a case study on prey DNA in Antarctic krill stomachs. Front Zool 5:12. doi: CrossRefGoogle Scholar
  24. Zaidi RH, Jaal Z, Hawkes NJ, Hemingway J, Symondson WOC (1999) Can multiple-copy sequences of prey DNA be detected amongst the gut contents of invertebrate predators? Mol Ecol 8:2081–2087. doi: CrossRefGoogle Scholar

Copyright information

© The Author(s) 2008

Open AccessThis is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License (, which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Authors and Affiliations

  • Christofer Troedsson
    • 1
    • 2
    Email author
  • Paolo Simonelli
    • 1
  • Verena Nägele
    • 2
  • Jens C. Nejstgaard
    • 3
  • Marc E. Frischer
    • 2
  1. 1.Department of BiologyUniversity of BergenBergenNorway
  2. 2.Skidaway Institute of OceanographySavannahGeorgia
  3. 3.Department of BiologyUNIFOBBergenNorway

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