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Many omnivorous arthropods act as important pest control agents in agroecosystems. Assessing their prey choices helps to better understand their biocontrol potential in different environmental settings. To this end, multi‐target approaches of molecular gut content analysis allow studying their prey choices. Yet, DNA‐based analysis of trophic interactions is challenged by the question of whether the detected food DNA was ingested primarily as direct prey, or secondarily via the gut content of a consumed animal. Experimental data are needed to understand the potential bias derived through secondary predation. Here we address this issue for a phloem‐feeding aphid consumed by omnivorous carabids. In feeding experiments, Pseudoophonus rufipes beetles were offered grain aphids Sitobion avenae as primary prey, which had fed on barley seedlings. Gut content samples of carabids collected after 0, 3, 9, 12, 24, 32 or 64 h of digestion time and whole‐body aphids were screened for the presence of aphid and plant DNA. Samples positive for plant DNA were subsequently tested with Poaceae‐specific primers. None of the 7.8% gut content samples that tested positive for general plant DNA amplified with the Poaceae primers. Moreover, none of the whole‐body extracted aphids tested positive for plant DNA, neither with general nor with family‐specific primers. We, therefore, conclude that the plant DNA detections in our experiment can be ascribed to remnants of plant material in the guts of the field‐collected carabids. Our findings demonstrate that these phloem‐feeding aphids are an unlikely cause of false trophic link assignment because no secondarily consumed plant DNA, derived either from aphid gut content or host‐plant environmental DNA on aphid bodies, could be detected in carabid gut content samples. [ABSTRACT FROM AUTHOR]

Fish are both consumers and prey, and as such part of a dynamic trophic network. Measuring how they are trophically linked, both directly and indirectly, to other species is vital to comprehend the mechanisms driving alterations in fish communities in space and time. Moreover, this knowledge also helps to understand how fish communities respond to environmental change and delivers important information for implementing management of fish stocks. DNA‐based methods have significantly widened our ability to assess trophic interactions in both marine and freshwater systems and they possess a range of advantages over other approaches in diet analysis. In this review we provide an overview of different DNA‐based methods that have been used to assess trophic interactions of fish as consumers and prey. We consider the practicalities and limitations, and emphasize critical aspects when analysing molecular derived trophic data. We exemplify how molecular techniques have been employed to unravel food web interactions involving fish as consumers and prey. In addition to the exciting opportunities DNA‐based approaches offer, we identify current challenges and future prospects for assessing fish food webs where DNA‐based approaches will play an important role. [ABSTRACT FROM AUTHOR]

Molecular techniques have become an important tool to empirically assess feeding interactions. The increased usage of next-generation sequencing approaches has stressed the need of fast DNA extraction that does not compromise DNA quality. Dietary samples here pose a particular challenge, as these demand high-quality DNA extraction procedures for obtaining the minute quantities of short-fragmented food DNA. Automatic high-throughput procedures significantly decrease time and costs and allow for standardization of extracting total DNA. However, these approaches have not yet been evaluated for dietary samples. We tested the efficiency of an automatic DNA extraction platform and a traditional CTAB protocol, employing a variety of dietary samples including invertebrate whole-body extracts as well as invertebrate and vertebrate gut content samples and feces. Extraction efficacy was quantified using the proportions of successful PCR amplifications of both total and prey DNA, and cost was estimated in terms of time and material expense. For extraction of total DNA, the automated platform performed better for both invertebrate and vertebrate samples. This was also true for prey detection in vertebrate samples. For the dietary analysis in invertebrates, there is still room for improvement when using the high-throughput system for optimal DNA yields. Overall, the automated DNA extraction system turned out as a promising alternative to labor-intensive, low-throughput manual extraction methods such as CTAB. It is opening up the opportunity for an extensive use of this cost-efficient and innovative methodology at low contamination risk also in trophic ecology. [ABSTRACT FROM AUTHOR]

1. Studies of the impact of predator diversity on biological pest control have shown idiosyncratic results. This is often assumed to be as a result of differences among systems in the importance of predator-predator interactions such as facilitation and intraguild predation. The frequency of such interactions may be altered by prey availability and structural complexity. A direct assessment of interactions among predators is needed for a better understanding of the mechanisms affecting prey abundance by complex predator communities. 2. In a field cage experiment, the effect of increased predator diversity (single species vs. three-species assemblage) and the presence of weeds (providing structural complexity) on the biological control of cereal aphids were tested and the mechanisms involved were investigated using molecular gut content analysis. 3. The impact of the three-predator species assemblages of aphid populations was found to be similar to those of the single-predator species treatments, and the presence or absence of weeds did not alter the patterns observed. This suggests that both predator facilitation and intraguild predation were absent or weak in this system, or that these interactions had counteracting effects on prey suppression. Molecular gut content analysis of predators provided little evidence for the latter hypothesis: predator facilitation was not detected and intraguild predation occurred at a low frequency. 4. The present study suggests additive effects of predators and, therefore, that predator diversity per se neither strengthens nor weakens the biological control of aphids in this system. [ABSTRACT FROM AUTHOR]

Soil-dwelling pests inflict considerable economic damage in agriculture but are hard to control. A promising strategy to reduce pest pressure on crops is to increase the plant diversity in agroecosystems. This approach, however, demands a sound understanding of species' interactions, which is widely lacking for subterranean herbivore-plant systems. Here, we examine the effects of plant diversification on wireworms, the soil-dwelling larvae of click beetles that threaten crops worldwide. We conducted a field experiment employing plant diversification by adding either wheat or a mix of six associated plants (grasses, legumes, and forbs) between rows of maize to protect it from Agriotes wireworms. Wireworm feeding behavior, dispersal between crop and associated plants, as well as maize damage and yield were examined. The former was assessed combining molecular gut content and stable isotope analysis. The pests were strongly attracted by the associated plants in August, when the crop was most vulnerable, whereas in September, shortly before harvest, this effect occurred only in the plant mix. In maize monoculture, the larvae stayed in the principal crop throughout the season. Larval d13C signatures revealed that maize feeding was reduced up to sevenfold in wireworms of the vegetationally diversified treatments compared to those of the maize monoculture. These findings were confirmed by molecular analysis, which additionally showed a dietary preference of wireworms for specific plants in the associated plant mix. Compared to the monoculture, maize damage was reduced by 38% and 55% in the wheat and plant mix treatment, which translated into a yield increase of 30% and 38%, respectively. The present findings demonstrate that increasing the plant diversity in agroecosystems provides an effective insurance against soil pests. The underlying mechanisms are the diversion of the pest from the principle crop and a changed feeding behavior. The deployment of diverse mixes of associated plants, tailored to the specific preferences of the soil herbivores, provides a promising strategy for managing subterranean pests while maintaining crop yield. [ABSTRACT FROM AUTHOR]

DNA-based gut content analysis has become an important tool for unravelling feeding interactions in invertebrate communities under natural conditions. It usually implies killing of the consumer and extracting the DNA from its food, using either the whole animal or its dissected gut. This post-mortem approach, however, is not suitable for investigating the diet of rare or protected species and also prohibits tracking individual dietary preferences as each consumer can provide trophic information only once. Moreover, removing large numbers of consumers from a habitat for analysis might critically change population densities and affect species interactions. Here, we present DNA-based analysis of invertebrate regurgitates, a novel approach to overcome these limitations. Conducting feeding experiments where adult Poecilus cupreus (Coleoptera: Carabidae) were fed with larvae of Amphimallon solstitiale (Coleoptera: Scarabaeidae), we show that detection success in regurgitates compared to samples prepared from whole beetles was similar or significantly enhanced for small/medium and large prey DNA fragments, respectively. Prey DNA detection success remained high in regurgitates stored in ethanol for 21 months at room temperature prior to DNA extraction. We conclude that in those invertebrates where regurgitates can be obtained, examination of food DNA in regurgitates offers many advantages over conventional post-mortem gut content analysis. [ABSTRACT FROM AUTHOR]

Abstract: Although a significant proportion of plant tissue is located in roots and other below-ground parts of plants, little is known on the dietary choices of root-feeding insects. This is caused by a lack of adequate methodology which would allow tracking below-ground trophic interactions between insects and plants. Here, we present a DNA-based approach to examine this relationship. Feeding experiments were established where either wheat (Triticum aestivum) or maize (Zea mays) was fed to Agriotes larvae (Coleoptera: Elateridae), allowing them to digest for up to 72 h. Due to the very small amount of plant tissue ingested (max = 6.76 mg), DNA extraction procedures and the sensitivity of polymerase chain reaction (PCR) had to be optimized. Whole-body DNA extracts of larvae were tested for the presence of both rbcL and trnL plastid DNA using universal primers. Moreover, based on cpDNA sequences encoding chloroplast tRNA for leucine (trnL), specific primers for maize and wheat were developed. With both, general and specific primers, plant DNA was detectable in the guts of Agriotes larvae for up to 72 h post-feeding, the maximum time of digestion in these experiments. No significant effect of time since feeding on plant DNA detection success was observed, except for the specific primers in maize-fed larvae. Here, plant DNA detection was negatively correlated with the duration of digestion. Both, meal size and initial mass of the individual larvae did not affect the rate of larvae testing positive for plant DNA. The outcomes of this study represent a first step towards a specific analysis of the dietary choices of soil-living herbivores to further increase our understanding of animal–plant feeding interactions in the soil. [ABSTRACT FROM AUTHOR]

Abstract: Molecular gut content analysis provides a highly specific and sensitive tool to examine the diet of soil invertebrates. Here, we present new polymerase chain reaction (PCR) assays for the detection of twelve prey taxa common in Central European forest soils. The assays target five species of collembolans as well as dipterans, gamasid and oribatid mites, lithobiid centipedes, spiders, staphylinid beetles and woodlice at the group level, amplifying 123–299 bp long DNA fragments. Cross-reactivity tests against 119 soil invertebrate taxa confirm their specificity. These new PCR assays were found to be highly sensitive, revealing the consumption of five different prey taxa in field-collected centipedes. Thus they provide a ready-to-use approach for unravelling trophic interactions among soil arthropods. [Copyright &y& Elsevier]