Quantification of copepod gut content by differential length amplification quantitative PCR (dla-qPCR)
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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.
KeywordsCalanoid 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.
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Open AccessThis is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License (https://doi.org/creativecommons.org/licenses/by-nc/2.0), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.