The analysis of food webs and their dynamics facilitates understanding of the mechanistic processes behind community ecology and ecosystem functions. Having accurate techniques for determining dietary ranges and components is critical for this endeavour. While visual analyses and early molecular approaches are highly labour intensive and often lack resolution, recent DNA-based approaches potentially provide more accurate methods for dietary studies. A suite of approaches have been used based on the identification of consumed species by characterization of DNA present in gut or faecal samples. In one approach, a standardized DNA region (DNA barcode) is PCR amplified, amplicons are sequenced and then compared to a reference database for identification. Initially, this involved sequencing clones from PCR products, and studies were limited in scale because of the costs and effort required. The recent development of next generation sequencing (NGS) has made this approach much more powerful, by allowing the direct characterization of dozens of samples with several thousand sequences per PCR product, and has the potential to reveal many consumed species simultaneously (DNA metabarcoding). Continual improvement of NGS technologies, on-going decreases in costs and current massive expansion of reference databases make this approach promising. Here we review the power and pitfalls of NGS diet methods. We present the critical factors to take into account when choosing or designing a suitable barcode. Then, we consider both technical and analytical aspects of NGS diet studies. Finally, we discuss the validation of data accuracy including the viability of producing quantitative data.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-294X.2011.05403.x

Who is eating what: diet assessment using next generation sequencing.

Molecular analysis of predation, through polymerase chain reaction amplification of prey remains within the faeces or digestive systems of predators, is a rapidly growing field, impeded by a lack of readily accessible advice on best practice. Here, we review the techniques used to date and provide guidelines accessible to those new to this field or from a different molecular biology background. Optimization begins with field collection, sample preservation, predator dissection and DNA extraction techniques, all designed to ensure good quality, uncontaminated DNA from semidigested samples. The advantages of nuclear vs. mitochondrial DNA as primer targets are reviewed, along with choice of genes and advice on primer design to maximize specificity and detection periods following ingestion of the prey by the predators. Primer and assay optimization are discussed, including cross-amplification tests and calibratory feeding experiments. Once primers have been made, the screening of field samples must guard against (through appropriate controls) cross contamination. Multiplex polymerase chain reactions provide a means of screening for many different species simultaneously. We discuss visualization of amplicons on gels, with and without incorporation of fluorescent primers. In more specialized areas, we examine the utility of temperature and denaturing gradient gel electrophoresis to examine responses of predators to prey diversity, and review the potential of quantitative polymerase chain reaction systems to quantify predation. Alternative routes by which prey DNA might get into the guts of a predator (scavenging, secondary predation) are highlighted. We look ahead to new technologies, including microarrays and pyrosequencing, which might one day be applied to this field.

Molecular analysis of predation: a review of best practice for DNA-based approaches.

5. Food Relationships

Food webs in agricultural systems are complex and trophic linkages are difficult to track using conventional methodologies. Here, we review three alternative approaches that allow empirical assessment of feeding interactions: DNA-based techniques, and stable isotope and fatty acid analyses. DNA-based methods, namely multiplex PCR and next-generation sequencing, allow identification of food types and host–parasitoid linkages, resulting in taxonomically highly resolved feeding networks. Stable isotopes and fatty acids reflect the assimilation of broader categories of resources, as metabolised into the consumers’ tissue, together with the associated energy and nutrient fluxes in the food web. We discuss the strengths of the approaches but also highlight their limitations, providing practical advice on which technique is best suited to answer specific questions in examining food web interactions in agroecosystems. Future refinements of these techniques, especially when used in combination, could herald a new era in agricultural food web ecology, enabling management and environmental impact to be placed in the mechanistic context of trophic networks.

Chapter Three – Empirically Characterising Trophic Networks: What Emerging DNA-Based Methods, Stable Isotope and Fatty Acid Analyses Can Offer

Following a request from EFSA, the Panel on Plant Protection Products and their Residues developed an opinion on the science behind the risk assessment of plant protection products for in-soil organisms. The current risk assessment scheme is reviewed, taking into account new regulatory frameworks and scientific developments. Proposals are made for specific protection goals for in-soil organisms being key drivers for relevant ecosystem services in agricultural landscapes such as nutrient cycling, soil structure, pest control and biodiversity. Considering the time-scales and biological processes related to the dispersal of the majority of in-soil organisms compared to terrestrial non-target arthropods living above soil, the Panel proposes that in-soil environmental risk assessments are made at in- and off-field scale considering field boundary levels. A new testing strategy which takes into account the relevant exposure routes for in-soil organisms and the potential direct and indirect effects is proposed. In order to address species recovery and long-term impacts of PPPs, the use of population models is also proposed.

Scientific Opinion addressing the state of the science on risk assessment of plant protection products for in-soil organisms

Lycosid spiders and alternative food: Feeding behavior and implications for biological control

Tracking aphid predation by lycosid spiders in spring-sown cereals using PCR-based gut-content analysis

Gut content analysis is a useful tool when studying arthropod predator-prey interactions. We used polymerase chain reaction (PCR) technique to examine how detection of prey DNA in the gut content of predators was influenced by digestion time and temperature. Such knowledge is critical before applying PCR-based gut content analysis to field collected predators. Larvae of the two-spotted ladybeetle (Adalia bipunctata L.) were fed with the bird cherry-oat aphid (Rhopalosiphum padi L.) at either 21°C or 14°C. After consuming one aphid, the predators were allowed to digest the prey for a range of time periods up to 24 hours. The influence of temperature on A. bipunctata feeding behavior was also recorded. From the fed larvae, total DNA was extracted and PCR reactions with R. padi specific primers were run. The number of A. bipunctata that tested positive for R. padi DNA was negatively related to the length of digestion time. Temperature influenced larval feeding behavior but did not have a significant effect on R. padi DNA detection. After pooling the data from both temperature treatments we estimated the time point when R. padi DNA could be amplified from 50% of the fed A. bipunctata by PCR to be 4.87 hours. With such a rapid decrease in prey DNA detection success, positive PCR reactions will most likely be the result of predation events occurring shortly before capture. If a defined digestion temperature range has proven not to influence prey detection, PCR data obtained from predators collected within that particular range can be interpreted in the same way.

The influence of time and temperature on molecular gut content analysis: Adalia bipunctata fed with Rhopalosiphum padi

Generalist arthropod predators are potential drivers of population dynamics in a wide variety of ecosystems but their feeding habits are often difficult to reveal as they are small, mobile, and live among dense vegetation or in soils. DNA-based gut-content analysis is a powerful tool that enables studies on arthropod predator–prey interactions. We studied lycosid spiders (Pardosa spp.) in agroecosystems to see if they consumed cereal aphids (Rhopalosiphum padi) and Collembolans at random, i.e., in proportion to their abundance in the field. We also tested if consumption of the target prey items was affected by the presence of alternative food. Spiders were captured in farmers’ fields and their gut-contents screened by PCR with R. padi and Collembola primers. On all sampling occasions, concurrent assessments of total prey availability were carried out. Spider predation rates on R. padi always exceeded 50 %. Spiders also tested positive for Collembola but to a lower and more varying degree. In general, Pardosa did not consume R. padi and Collembolans in relation to their abundance in the field. Aphid predation was much higher than expected whereas consumption of Collembolans was considerably lower. The presence of alternative prey influenced consumption of the aphid. It was concluded that prey consumption by Pardosa spiders generally cannot be assumed to simply mirror prey availability. The spatial distribution of the target prey needs to be considered as well as the abundance, composition, and nutritional content of potential alternative food items.

Feeding habits of lycosid spiders in field habitats

Flea beetles, Phyllotreta Chevrolat (Coleoptera: Chrysomelidae) species, are often found in oilseed rape (OSR), Brassica napus Linnaeus (Brassicaceae). Among predators in the generalist predator complex present in agricultural fields, wolf spiders (Araneae: Lycosidae) are found on the ground and cobweb spiders (Araneae: Theridiidae) build webs in the foliage. We present group-specific primers developed for five flea beetle species within the genus Phyllotreta and study the incidence of predation of flea beetles by these spider groups using DNA-based gut-content analysis. Wolf spiders of the genus Pardosa Koch and the cobweb spider, Phylloneta impressa (Koch), were collected in three winter OSR fields. Flea beetle densities as well as the occurrence of predators and alternative prey were monitored. In total 19.4% of the collected Pardosa tested positive for flea beetle DNA in the polymerase chain reaction analyses, whereas 10% P. impressa were positive. Pardosa were more likely to be positive for flea beetle DNA when Pardosa activity density was low. Phylloneta impressa were more likely to be positive for flea beetle DNA if they were positive for pollen beetle DNA. Implications of these results for conservation biological control and future studies of food webs in OSR are discussed.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S0008347X14000145

Anna-Karin Kuusk

Papers

Lycosid spiders and alternative food: Feeding behavior and implications for biological control

Tracking aphid predation by lycosid spiders in spring-sown cereals using PCR-based gut-content analysis

The influence of time and temperature on molecular gut content analysis: Adalia bipunctata fed with Rhopalosiphum padi

Feeding habits of lycosid spiders in field habitats

Consumption of flea beetles (Phyllotreta, Coleoptera: Chrysomelidae) by spiders in field habitats detected by molecular analysis