Rapid screening of invertebrate predators for multiple prey DNA targets

Abstract

DNA-based techniques are providing valuable new approaches to tracking predator-prey interactions. The gut contents of invertebrate predators can be analysed using species-specific primers to amplify prey DNA to confirm trophic links. The problem is that each predator needs to be analysed with primers for the tens of potential prey available at a field site, even though the mean number of species detected in each gut may be as few as one or two. Conducting all these PCRs (polymerase chain reactions) is a lengthy process, and effectively precludes the analysis of the hundreds of predators that might be required for a meaningful ecological study. We report a rapid, more sensitive and practical approach. Multiplex PCRs, incorporating fluorescent markers, were found to be effective at amplifying degraded DNA from predators' guts and could amplify mitochondrial DNA fragments from 10+ species simultaneously without 'drop outs'. The combined PCR products were then separated by size on polyacrylamide gels on an ABI377 sequencer. New primers to detect the remains of aphids, earthworms, weevils and molluscs in the guts of carabid predators were developed and characterized. The multiplex-sequencer approach was then applied to field-caught beetles, some of which contained DNA from as many as four different prey at once. The main prey detected in the beetles proved to be earthworms and molluscs, although aphids and weevils were also consumed. The potential of this system for use in food-web research is discussed.

Faeces of generalist predators as ‘biodiversity capsules’: A new tool for biodiversity assessment in remote and inaccessible habitats

“…Although this is not an issue for species with highly specialised diets (Rougerie et al, 2011), this is particularly limiting for generalist predators that feed on a variety of prey species. Until recently, such mixed DNA samples could be analysed only after the development of large panels of species-specific primers (Jarman et al, 2004) used in complex multiplex PCRs (Harper et al, 2005;King et al, 2011) or cloning analyses (Zeale et al, 2011).…”

Detecting arthropod intraguild predation in the field

“…As a consequence, PCR primers might not bind in all sampled individuals, and so prey detection might fail. High levels of intra-specific variability among lumbricid earthworms (Aporrectodea caliginosa (Savigny), A. longa (Ude), A. rosea (Savigny), Allolobophora chlorotica (Savigny), Lumbricus castaneus (Savigny), L. festivus (Savigny), L. rubellus Hoffmeister, L. terrestris L. and Octolasion cyaneum (Savigny)) foiled attempts by Harper et al (2005) to design COI specific primers in a study investigating spider prey.…”

“…Chen et al 2000;McMillan et al 2007), cytochrome b (Pons 2006) and the mitochondrial (12S or 16S) ribosomal genes (e.g. Harper et al 2005). There have also been a few studies using target sequences within multi-copy nuclear ribosomal genes.…”

“…to include in a single PCR reaction multiple primer pairs designed to detect different prey species. A multiplex approach was developed by Harper et al (2005), who recognised that certain kinds of food web studies needed a PCR based system that could simultaneously screen a generalist predator for a range of prey species. As standard agarose gel electrophoresis cannot resolve multiple short fragments, the analysis of multiplex PCR products requires the use of detection systems discriminating fragments amplified with primer pairs labeled with fluorescent markers which can substantially add to the experimental costs.…”

“…RT-PCR is cost effective when the presence of many target sequences can be assessed in a single PCR reaction. Thus, it is possible to detect multiple prey types in a single analysis using a multiplex PCR (Harper et al 2005). This is also the case with GC-MS techniques, if retention times and/or mass spectra of each compound are sufficiently different (Sloggett et al 2009;Hautier et al 2011).…”