PCR and quantitative-PCR to analyse prey-specific cladocerans feeding: optimisation of Planktothrix detection in Daphnia diet

Cladocerans play a central role in lacustrine food webs as grazers of plankton and as prey for many planktivorous predators. Thus, the understanding of their trophic interactions is critical and emphasises the need for the development of new tools to investigate zooplankton trophic processes. In this respect, a promising strategy for assessment of feeding of metazooplankton is the use of prey specific nucleic acid molecules as biomarkers of trophic transfer, as recently developed in marine systems for copepods (Nejstgaard et al 2008, Troedsson et al 2009). In this study, we focused more especially on the DNA-based detection of toxic cyanobacteria (Planktothrix rubescens) in Daphnia diet. This cladoceran represents an essential food resource for young perch (Perca fluviatilis) and whitefish (Coregonus lavaretus) in the mesotrophic Lake Bourget which is characterized by the recurrent presence of Planktothrix rubescens. Consequently, these cladocerans represent a potential way of cyanotoxins transfer to the planktivorous fish. The molecular gut content analysis of Daphnia could be a powerful tool to investigate in situ the importance of this potential pathway. In this study, we developed a DNA-based method to detect Planktothrix in cladocerans feeding by molecular approaches. To develop the assay, we conducted controlled laboratory feeding experiments using phytoplanktonic cultures and Daphnia clones initially isolated from Lake Bourget. Several methodological approaches were evaluated to extract total DNA from Daphnia and to define the minimum number of Daphnia suitable for this molecular tracking. In parallel, we tested and validated the specificity of primers for Planktothrix detection and quantification by PCR and qPCR (TaqMan and SybrGreen). We present the first results of successful extraction, molecular detection and quantification of a specific prey consumed by Daphnia. Using Daphnia hyalina fed either with P. rubescens alone or with an equal mix of Scenedesmus and P. rubescens, as model systems, DNA originating from P. rubescens was unambiguously detected and quantified in DNA extracts from cladocerans. The results suggest that the quantification of prey items by qPCR could be used to estimate the feeding rates. Future studies under controlled laboratory and field conditions should be conducted to correct for breakdown in prey DNA and perform extensive calibrations in order to achieve a quantitative measure of feeding rates in situ.