PCR-DHPLC assay for the identification of predator-prey interactions

Abstract

Denaturing high-performance liquid chromatography (DHPLC) is a relatively new method for separating amplicons in a mixture, and was recently developed for parasite detection in the blue crab Callinectes sapidus. That assay used a peptide nucleic acid (PNA) PCR hybridization blocking probe (PNA–PCR–DHPLC) to decrease the generic PCR bias of dominant templates (the host) in the mixture prior to separation on the DHPLC column, thus enhancing the less abundant parasite DNA. The same assay and rational can be used to investigate predator–prey interactions. However, in ecosystem studies with many predator–prey relationships, development of specific PNA-blocking probes for each predator would be too laborious. Here, we have developed a PCR–DHPLC assay excluding the dominant predator amplicons in a first DHPLC run, followed by re-amplification of the non-predator retention volumes and further separation and characterization in a second DHPLC run. This assay generated data on the specific trophic interactions between the calanoid copepod Limnocalanus macrurus and its prey from a seasonal sampling programme. The assay provides an efficient way for an unbiased screening of predator–prey relationships, and although developed for L. macrurus in this study, the approach has wide applicability for any predator–prey interaction.