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.

Understanding species’ interactions and the robustness of interaction networks to species loss is essential to understand the effects of species’ declines and extinctions. In most studies, different types of networks (such as food webs, parasitoid webs, seed dispersal networks, and pollination networks) have been studied separately. We sampled such multiple networks simultaneously in an agroecosystem. We show that the networks varied in their robustness; networks including pollinators appeared to be particularly fragile. We show that, overall, networks did not strongly covary in their robustness, which suggests that ecological restoration (for example, through agri-environment schemes) benefitting one functional group will not inevitably benefit others. Some individual plant species were disproportionately well linked to many other species. This type of information can be used in restoration management, because it identifies the plant taxa that can potentially lead to disproportionate gains in biodiversity.

/pdf/the-robustness-and-restoration-of-a-network-of-ecological-l1uol14jgi.pdf

The robustness and restoration of a network of ecological networks.

When natural habitat fails to enhance biological pest control – Five hypotheses ☆

At the end of the class, students will be able to answer the following questions: • What is organic agriculture? • Where do I go to get certification in organic agriculture? • How can I get more information about organic agriculture? What is organic? • The term " organic " is not synonymous to the terms " natural " or " eco-friendly. " • The label " natural " on foodstuff does not guarantee complete adherence to organic practices as defined by a law. • Produce food of high quality in sufficient quantity • Maintain biological diversity within the farming system • Maintain long-term soil fertility • Rely on renewable resources in locally organized agricultural systems • Minimize pollution and protect the environment What is allowed in organic crop production? • New varieties of crops and agricultural technologies • Crop rotations, cover crops and natural-based products that maintain soil fertility • Biological, cultural and physical methods to limit pest expansion and increase population of beneficial insects

英国有机农业（Organic farming）的新举措

Digital Polymerase Chain Reaction (dPCR) is a novel method for the absolute quantification of target nucleic acids. Quantification by dPCR hinges on the fact that the random distribution of molecules in many partitions follows a Poisson distribution. Each partition acts as an individual PCR microreactor and partitions containing amplified target sequences are detected by fluorescence. The proportion of PCR-positive partitions suffices to determine the concentration of the target sequence without a need for calibration. Advances in microfluidics enabled the current revolution of digital quantification by providing efficient partitioning methods. In this review, we compare the fundamental concepts behind the quantification of nucleic acids by dPCR and quantitative real-time PCR (qPCR). We detail the underlying statistics of dPCR and explain how it defines its precision and performance metrics. We review the different microfluidic digital PCR formats, present their underlying physical principles, and analyze the technological evolution of dPCR platforms. We present the novel multiplexing strategies enabled by dPCR and examine how isothermal amplification could be an alternative to PCR in digital assays. Finally, we determine whether the theoretical advantages of dPCR over qPCR hold true by perusing studies that directly compare assays implemented with both methods.

dPCR: A Technology Review.

1. Multiplex PCR is a valuable tool in many biological studies but it is a multifaceted procedure that has to be planned and optimised thoroughly to achieve robust and meaningful results. In particular, primer concentrations have to be adjusted to assure an even amplification of all targeted DNA fragments. Until now, total DNA extracts were used for balancing primer efficiencies; however, the applicability for comparisons between taxa or different multiple-copy genes was limited owing to the unknown number of template molecules present per total DNA.

2. Based on a multiplex system developed to track trophic interactions in high Alpine arthropods, we demonstrate a fast and easy way of generating standardised DNA templates. These were then used to balance the amplification success for the different targets and to subsequently determine the sensitivity of each primer pair in the multiplex PCR.

3. In the current multiplex assay, this approach led to an even amplification success for all seven targeted DNA fragments. Using this balanced multiplex PCR, methodological bias owing to variation in primer efficiency will be avoided when analysing field-derived samples.

4. The approach outlined here allows comparing multiplex PCR sensitivity, independent of the investigated species, genome size or the targeted genes. The application of standardised DNA templates not only makes it possible to optimise primer efficiency within a given multiplex PCR, but it also offers to adjust and/or to compare the sensitivity between different assays. Along with other factors that influence the success of multiplex reactions, and which we discuss here in relation to the presented detection system, the adoption of this approach will allow for direct comparison of multiplex PCR data between systems and studies, enhancing the utility of this assay type.

https://besjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/j.2041-210X.2012.00215.x

Advances in multiplex PCR: balancing primer efficiencies and improving detection success

Generalist predators and parasitoids are considered to be important regulators of aphids. The former not only feed on these pests, but might also consume parasitoids at all stages of development. This direct or coincidental interference affects the natural control of aphids, the scale of which is largely unknown, and it has rarely been examined under natural conditions. Here, molecular diagnostics were used to track trophic interactions in an aphid-parasitoid-generalist predator community during the build-up of a cereal aphid population. We found that generalist predators, principally carabid and staphylinid beetles as well as linyphiid spiders, had strong trophic links to both parasitoids and aphids. Remarkably, more than 50% of the parasitoid DNA detected in predators stems from direct predation on adult parasitoids. The data also suggest that coincidental intraguild predation is common too. Generalist predators, hence, disrupt parasitoid aphid control, although the levels at which the predators feed on pests and parasitoids seem to vary significantly between predator taxa. Our results suggest that taxon-specific trophic interactions between natural enemies need to be considered to obtain a more complete understanding of the route to effective conservation biological control.

/pdf/generalist-predators-disrupt-parasitoid-aphid-control-by-2me7i6aeoh.pdf

Generalist predators disrupt parasitoid aphid control by direct and coincidental intraguild predation.

Parasitoid control of aphids in organic and conventional farming systems

Molecular methods have become an important tool for studying feeding interactions under natural conditions. Despite their growing importance, many methodological aspects have not yet been evaluated but need to be considered to fully exploit the potential of this approach. Using feeding experiments with high alpine carabid beetles and lycosid spiders, we investigated how PCR annealing temperature affects prey DNA detection success and how post-PCR visualization methods differ in their sensitivity. Moreover, the replicability of prey DNA detection among individual PCR assays was tested using beetles and spiders that had digested their prey for extended times postfeeding. By screening all predators for three differently sized prey DNA fragments (range 116–612 bp), we found that only in the longest PCR product, a marked decrease in prey detection success occurred. Lowering maximum annealing temperatures by 4 � Cr esulted in signif icantly increased prey DNA detection rates in both predator taxa. Among the three post-PCR visualization methods, an eightfold difference in sensitivity was observed. Repeated screening of predators increased the total number of samples scoring positive, although the proportion of samples testing positive did not vary significantly between different PCRs. The present findings demonstrate that assay sensitivity, in combination with other methodological factors, plays a crucial role to obtain robust trophic interaction data. Future work employing molecular prey detection should thus consider and minimize the methodologically induced variation that would also allow for better cross-study comparisons.

Optimizing methods for PCR-based analysis of predation.

Pioneer communities establishing themselves in the barren terrain in front of glacier forelands consist principally of predator species such as carabid beetles and lycosid spiders. The fact that so many different predators can co-inhabit an area with no apparent primary production was initially explained by allochthonous material deposited in these forelands. However, whether these populations can be sustained on allochthonous material alone is questionable and recent studies point towards this assumption to be flawed. Intraguild predation (IGP) might play an important role in these pioneer predator assemblages, especially in the very early successional stages where other prey is scarce. Here, we investigated IGP between the main predator species and their consumption of Collembola, an important autochthonous alternative prey, within a glacier foreland in the Otztal (Austrian Alps). Multiplex PCR and stable isotope analysis were used to characterize the trophic niches in an early and late pioneer stage over 2 years. Results showed that intraguild prey was consumed by all invertebrate predators, particularly the larger carabid species. Contrary to our initial hypothesis, the DNA detection frequency of IGP prey was not significantly higher in early than in late pioneer stage, which was corroborated by the stable isotope analysis. Collembola were the most frequently detected prey in all of the predators, and the overall prey DNA detection patterns were consistent between years. Our findings show that IGP appears as a constant in these pioneer predator communities and that it remains unaffected by successional changes.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/mec.12649

Lorna Raso

Papers

Advances in multiplex PCR: balancing primer efficiencies and improving detection success

Generalist predators disrupt parasitoid aphid control by direct and coincidental intraguild predation.

Parasitoid control of aphids in organic and conventional farming systems

Optimizing methods for PCR-based analysis of predation.

Intraguild predation in pioneer predator communities of alpine glacier forelands