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Next‐generation sequencing (NGS) is increasingly used for diet analyses; however, it may not always describe diet samples well. A reason for this is that diet samples contain mixtures of food DNA in different amounts as well as consumer DNA which can reduce the food DNA characterized. Because of this, detections will depend on the relative amount and identity of each type of DNA. For such samples, diagnostic PCR will most likely give more reliable results, as detection probability is only marginally dependent on other copresent DNA. We investigated the reliability of each method to test (a) whether predatory beetle regurgitates, supposed to be low in consumer DNA, allow to retrieve prey sequences using general barcoding primers that co‐amplify the consumer DNA, and (b) to assess the sequencing depth or replication needed for NGS and diagnostic PCR to give stable results. When consumer DNA is co‐amplified, NGS is better suited to discover the range of possible prey, than for comparing co‐occurrences of diet species between samples, as retested samples were repeatedly different in prey detections with this approach. This shows that samples were incompletely described, as prey detected by diagnostic PCR frequently were missed by NGS. As the sequencing depth needed to reliably describe the diet in such samples becomes very high, the cost‐efficiency and reliability of diagnostic PCR make diagnostic PCR better suited for testing large sample‐sets. Especially if the targeted prey taxa are thought to be of ecological importance, as diagnostic PCR gave more nested and consistent results in repeated testing of the same sample. [ABSTRACT FROM AUTHOR]

The nucleotide variation in the cytochrome c oxidase subunit I (COI) gene makes it ideal for assigning sequences to species. However, this variability also makes it difficult to design truly universal primers. Here, we present the forward primer "Sauron‐S878," specifically designed to facilitate library preparation for metabarcoding. This primer is modified to improve the coverage of terrestrial species compared to the primer mCOIintF, optimized for aquatic systems, which raised the in silico coverage from 74.4% to 98.3% of available NCBI sequences (perfect match in 3′ region, up to three mismatches in remaining primer). When paired with the reverse primer "jgHCO2198" (fragment length ~313 bp), these primers amplified 98.4% of 255 tested DNA extracts from various taxa, which are better than many other common COI barcoding primers. Furthermore, a single‐tube protocol was developed, wherein these primers amplify the target gene, and attach MIDs and Illumina sequencing adapters in one reaction. This eliminates the need for re‐amplification or enzymatic ligation during library preparation while keeping the flexibility to modularly combine primers and MIDs. Using the single‐tube approach, three replicates of three mock samples were sequenced on a MiSeq platform with no adverse effects compared to commercial Nextera indexing kits. From this run, 75% of all included taxa could be recovered, with no considerable bias among taxonomic groups. Despite the fact that 98.4% of the extracts were confirmed to amplify in vitro, this number was lower than expected. A reason for this discrepancy was a clear link between the relative concentration of a specific DNA type in the template and the number of returned reads for this DNA. We would argue that such a bias may be especially problematic in metabarcoding where samples usually contain trace DNA in unknown amounts. However, how this affects the completeness of metabarcoding results has yet been poorly investigated. Here, we present the forward primer "Sauron‐S878," specifically designed to facilitate library preparation for metabarcoding. This primer exhibits little bias between taxonomic groups. Furthermore, when paired with the reverse primer "jgHCO2198," these primers can be used to attach MIDs and Illumina sequencing adapters in one reaction. [ABSTRACT FROM AUTHOR]

Abstract: The rapidly growing field of molecular diet analysis is becoming increasingly popular among ecologists, especially when investigating methodologically challenging groups, such as invertebrate generalist predators. Prey DNA detection success is known to be affected by multiple factors; however, the type of dietary sample has rarely been considered. Here, we address this knowledge gap by comparing prey DNA detection success from three types of dietary samples. In a controlled feeding experiment, using the carabid beetle Pterostichus melanarius as a model predator, we collected regurgitates, faeces and whole consumers (including their gut contents) at different time points postfeeding. All dietary samples were analysed using multiplex PCR, targeting three different length DNA fragments (128, 332 and 612 bp). Our results show that both the type of dietary sample and the size of the DNA fragment contribute to a significant part of the variation found in the detectability of prey DNA. Specifically, we observed that in both regurgitates and whole consumers, prey DNA was detectable significantly longer for all fragment sizes than for faeces. Based on these observations, we conclude that prey DNA detected from regurgitates and whole consumers DNA extracts are comparable, whereas prey DNA detected from faeces, though still sufficiently reliable for ecological studies, will not be directly comparable to the former. Therefore, regurgitates and faeces constitute a useful, nonlethal source for dietary information that could be applied to field studies in situations when invertebrate predators should not be killed. [ABSTRACT FROM AUTHOR]

• Landscape had a lower effect on aphids and their parasitoids than fertilisation. • Fertilisation affected plants positively. • Fertilisation affected aphids and primary parasitism rate negatively. • Fertilisation had no effect on secondary parasitism rate. • H 2 ' and generality between parasitoids are affected by landscape and fertilisation. Agricultural intensification can impact agrobiodiversity in several ways such as in terms of population densities, community composition and food web interactions across all trophic levels. This effect can be investigated at two scales: field-scale and landscape scale. Here it was assessed how the impact of fertilisation (within field) and landscape complexity (within landscape) impact cereal aphid-primary parasitoid-secondary parasitoid systems in winter wheat in Germany. A newly developed molecular technique was used to quantify species-specific linkages between aphids, primary parasitoids and secondary parasitoids sampled in fertilised and unfertilised plots in either simple or complex structured landscapes. The results show a stronger effect of fertilisation than landscape complexity on the groups: fertilisation positively affected the crop plants while it negatively affected both the density of the cereal aphid Sitobion avenae and its primary parasitism rates whereas no effect on the level of secondary parasitism rates was observed. Landscape complexity had no effect on plants, aphids, as well as on primary parasitism rate. In case of secondary parasitism rate there was an effect in interaction with sampling date. Field identity accounted for the strongest effect on parasitoid community composition (10.6% of the variance) from all tested variables, while fertilisation and landscape complexity had almost no effect (1.1% and no effect). Nevertheless, a weak cascading effect of both environmental factors could be observed as the primary-secondary parasitoid network structure responded to both. However, these observed effects on food webs strongly depended on species identity, highlighting the need of species-level food web assessment. [ABSTRACT FROM AUTHOR]