Quantification of copepod gut content by differential length amplification quantitative PCR (dla-qPCR)
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ORIGINAL PAPER
Quantification of copepod gut content by differential length amplification quantitative PCR (dla-qPCR) Christofer Troedsson Æ Paolo Simonelli Æ Verena Na¨gele Æ Jens C. Nejstgaard Æ Marc E. Frischer
Received: 26 March 2008 / Accepted: 20 October 2008 / Published online: 8 November 2008 Ó The Author(s) 2008. This article is published with open access at Springerlink.com
Abstract 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 Communicated by A. Atkinson. C. Troedsson (&) P. Simonelli Department of Biology, University of Bergen, P.O. Box 7800, 5020 Bergen, Norway e-mail: [email protected] C. Troedsson V. Na¨gele M. E. Frischer Skidaway Institute of Oceanography, 10 Ocean Science Circle, 31411 Savannah, Georgia J. C. Nejstgaard Department of Biology, UNIFOB, P.O. Box 7800, 5020 Bergen, Norway
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.
Introduction Mesozooplankton, in particular copepods, dominate the marine plankton biomass and play a critical role in the marine food web as selective predators, selective nutrient regenerators, carbon export regulators, and mediators of energy to higher trophic levels (Verity and Smetacek 1996). Thus, the ability to accurately estimate copepod feeding preferences and rates is vital for a quantitative understanding of trophic interactions and processes that structure marine ecosystems. However, because of methodological constraints, it remains a challenge to identify and quantify copepod feeding in situ without significant experimental bias (Ba˚mstedt et al. 2000; Nejstgaard et al. 2003; Nejstgaard et al. 2008). In view of these challenges,
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