White grubs (larvae of Coleoptera: Scarabaeidae) are abundant in below-ground systems and can cause considerable damage to a wide variety of crops by feeding on roots. White grub populations may be controlled by natural enemies, but the predator guild of the European species is barely known. Trophic interactions within soil food webs are difficult to study with conventional methods. Therefore, a polymerase chain reaction (PCR)-based approach was developed to investigate, for the first time, a soil insect predator-prey system. Can, however, highly sensitive detection methods identify carrion prey in predators, as has been shown for fresh prey? Fresh Melolontha melolontha (L.) larvae and 1- to 9-day-old carcasses were presented to Poecilus versicolor Sturm larvae. Mitochondrial cytochrome oxidase subunit I fragments of the prey, 175, 327 and 387 bp long, were detectable in 50% of the predators 32 h after feeding. Detectability decreased to 18% when a 585 bp sequence was amplified. Meal size and digestion capacity of individual predators had no influence on prey detection. Although prey consumption was negatively correlated with cadaver age, carrion prey could be detected by PCR as efficiently as fresh prey irrespective of carrion age. This is the first proof that PCR-based techniques are highly efficient and sensitive, both in fresh and carrion prey detection. Thus, if active predation has to be distinguished from scavenging, then additional approaches are needed to interpret the picture of prey choice derived by highly sensitive detection methods.

The analysis of prey DNA in faeces is a non-invasive approach to examine the diet of birds. However, it is poorly known how gut transition time, environmental factors and laboratory treatments such as storage conditions or DNA extraction procedures affect the detection success of prey DNA. Here, we examined several of these factors using faeces from carrion crows fed with insect larvae. Faeces produced between 30 min and 4 h post-feeding tested positive for insect DNA, representing the gut transition time. Prey detection was not only possible in fresh but also in 5-day-old faeces. The type of surface the faeces were placed on for these 5 days, however, affected prey DNA detection success: samples placed on soil provided the lowest rate of positives compared to faeces left on leaves, on branches and within plastic tubes. Exposing faeces to sunlight and rain significantly lowered prey DNA detection rates (17% and 68% positives in exposed and protected samples, respectively). Storing faeces in ethanol or in the freezer did not affect molecular prey detection. Extracting DNA directly from larger pieces of faecal pellets resulted in significantly higher prey detection rates than when using small amounts of homogenized faeces. A cetyltrimethyl ammonium bromide-based DNA extraction protocol yielded significantly higher DNA detection rates (60%) than three commercial kits, however, for small amounts of homogenized faeces only. Our results suggest that collecting faeces from smooth, clean and non-absorbing surfaces, protected from sunlight and rain, improves DNA detection success in avian faeces.

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Plant roots represent an important food source for soil-dwelling animals, but tracking herbivore food choices below-ground is difficult. Here, we present an optimized PCR assay for the detection of plant DNA in the guts of invertebrates, using general plant primers targeting the trnT-F chloroplast DNA region. Based on this assay, we assessed the influence of plant identity on the detectability of ingested plant DNA in Agriotes click beetle larvae. Six different plant species were fed to the insects, comprising a grass, a legume and four nonlegume forbs. Moreover, we examined whether it is possible to amplify DNA of decaying plants and if DNA of decayed plant food is detectable in the guts of the larvae. DNA of the ingested roots could be detected in the guts of the larvae for up to 72-h post-feeding, the maximum digestion time tested. When fed with living plants, DNA detection rates differed significantly between the plant species. This may be ascribed to differences in the amount of plant tissue consumed, root palatability, root morphology and/or secondary plant components. These findings indicate that plant identity can affect post-feeding DNA detection success, which needs to be considered for the interpretation of molecularly derived feeding rates on plants. Amplification of plant DNA from decaying plants was possible as long as any tissue could be retrieved from the soil. The consumption of decaying plant tissue could also be verified by our assay, but the insects seemed to prefer fresh roots over decaying plant material.