Quantitative PCR to estimate copepod feeding
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Copepods play a central role in marine food webs as grazers of plankton and as key prey for many predators. Therefore, quantifying their specific trophic interactions is critical for understanding the role of copepods in ocean processes. However, because of methodological constraints, it remains difficult to investigate in situ copepod feeding without reliance on laborious intrusive and potentially biased incubation approaches. Recent advances in PCR-based methodologies have demonstrated the feasibility of directly identifying copepod diets based on prey DNA sequences. Yet, obtaining quantitative information from these approaches remains challenging. This study presents results of systematic efforts to develop a quantitative PCR (qPCR) assay targeted to 18S rRNA gene fragments to estimate copepod gut content of specific species of prey algae. These results were first compared to gut content estimates based on fluorescence in the copepod Calanus finmarchicus fed monocultures of two different microalgae species in controlled laboratory studies. In subsequent field studies, we compared feeding rates obtained by microscopy and qPCR for Temora longicornis and Acartia clausi feeding on the haptophyte Phaeocystis globosa in natural blooms. These investigations demonstrate a semi-quantitative relationship between gut content estimates derived from qPCR, gut pigment, and direct microscopy. However, absolute estimates of gut content based on qPCR methodology were consistently lower than expected. This did not appear to be explained by the extraction methods used, or interference by non-target (predator) DNA in the PCR reactions, instead suggesting digestion of prey-specific nucleic acids. Furthermore, the 18S rDNA target gene copy number of the phytoplankton varied with growth phase. Nonetheless, when prey target gene copy number in the ambient water is quantified, the qPCR-approach can be compared to other methods, and then used to semi-quantitatively estimate relative copepod grazing on specific prey in situ without involving further incubations. A distinct advantage of a DNA-based molecular approach compared to gut fluorescence and direct microscopic observation, is the ability to detect non-pigmented and macerated prey. Future studies should aim to correct for breakdown in prey DNA and perform extensive calibrations to other methods in order to achieve a quantitative measure of feeding rates in situ.
KeywordsPrey Cell Natural Plankton Real Time qPCR Assay Phaeocystis Globosa Algal Prey
We would like to thank the crews of the R.V. “Côtes de la Manche” for assistance, Valérie Gentilhomme as the coordinator of both PNEC & CPER programs, Alexei Sentchev for field equipment, Natacha Guiselin for her help with phytoplankton analysis of field samples, and Dr. Elvire Antajan for assistance with copepods during the field study. We also thank the five anonymous reviewers for their valuable suggestions. The figures were prepared by Ms. Anna Boyette (SkIO). This work was supported by the Norwegian Research Council (NRC) project 152714/120 30 to JCN, NRC project 145326/432 to CT, and the U.S. National Science Foundation Office of Polar Programs grant (OPP-00-83381) and the US Department of Energy Biotechnology Investigations—Ocean Margins Program (FG02-98EF 62531) to MEF.
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