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DNA metabarcoding is increasingly important for studying feeding interactions, yet it remains unresolved whether reporting read counts or occurrences is to be preferred. To address this issue for gut content samples, basic experimental data on the relationship between read numbers and initial prey DNA amounts and how both change over digestion time is needed. Using regurgitates of the carabid Pseudoophonus rufipes the digestion of Taraxacum officinale seeds was documented for 128 h post-feeding to determine how the number of (1) seed DNA copies and (2) metabarcoding reads change over digestion time, and (3) how they correlate to each other. Additionally, we tested (4) whether PCR cycle-numbers during library preparation affect this correlation. The number of copies and reads both decreased with digestion time, but variation between samples was high. Read and copy numbers correlated when using a library preparation protocol with 35 cycles (R2 = 42.0%), yet a reduction to 30 cycles might significantly improve this correlation, as indicated by additional PCR testing. Our findings show that protocol optimization is important to reduce technical distortions of read numbers in metabarcoding analysis. However, high inter-sample variation, likely due to variable digestive efficiency of individual consumers, can blur the relationship between the amount of food consumed and metabarcoding read numbers. [ABSTRACT FROM AUTHOR]

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]

• Intraguild predation within beetle communities was analysed in cereal fields. • Organic fertilization increased non-pest prey and lowered intraguild predation. • Lower intraguild predation correlated with lower pest spread, but not density. • No effect of organic fertilization was found on pest density or consumption. • Fertilization effects may offset pest predation from lower predator interference. Intraguild predation is an extreme form of competition, that depends on resource availability, niche overlap, behaviour and diversity. However, despite modelling and mesocosm experiments, field studies are scarce, and we know nothing of its temporal dynamics within real-world food webs, particularly in the context of biological control. To address this, we added manure to cereal fields to increase decomposer prey, and analysed the diet of over 6000 predators over the entire cropping season during two consecutive years. Our results show that manure increased available prey temporarily and reduced intraguild predation. With this organic fertilization, a reduction in pest spread between tillers within fields correlated with lower intraguild predation, while pest density remained unchanged across treatments, making the net effect on biological control unclear. We hypothesize that the mechanism responsible for why lowering intraguild predation reduces pest spread are changes in predator behaviour, that allow predators to exert more pressure on pests. Conversely, the absence of an effect on pest density may be linked to other effects of fertilization, such as increased pest and plant growth counteracting any increase in predation pressure. The high temporal resolution of our data showed how dynamic IGP is throughout the season, which stresses the importance of studying food webs over time, to generate a mechanistic understanding of biological control. [ABSTRACT FROM AUTHOR]

DNA-based diet analysis of natural enemies is a valuable tool for unravelling the food choice of predators in agroecosystems. It enables the rapid identification of potential biocontrol agents of invertebrate pests. Here, we present a new multiplex PCR system for the identification of pest slug species in the diet of their natural enemies such as carabid beetles. It comprises three species-specific primers targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene to detect DNA of the common garden slug, Arion distinctus (Stylommatophora: Arionidae), the Iberian slug, Arion lusitanicus (Stylommatophora: Arionidae) and the grey field slug Deroceras reticulatum (Stylommatophora: Agriolimacidae). We also include (super)family-specific primers for Arionidae and Limacoidea, which amplify parts of the 28S gene for ribosomal RNA (rRNA) in order to identify a wider range of slugs. The amplicons for Arionidae can be assigned to a total of seven Central European slug species of this family and the amplicons for Limacoidea to ten species. The multiplex assay showed high specificity against DNA extracts of field-collected slugs and co-occurring invertebrates. The assay also exhibited high sensitivity, which was confirmed by testing it with 223 dietary samples from field-collected carabids as potential natural enemies of slugs in agroecosystems. This methodology represents a new, cost-effective, highly sensitive and specific approach for the identification of common Central European slug species as well as for analysing trophic interactions to identify natural enemies for further biological control development. It can also be applied in any study where a rapid and reliable identification of slugs is needed. [ABSTRACT FROM AUTHOR]

Intensified agriculture, a driver of biodiversity loss, can diminish ecosystem functions and their stability. Biodiversity can increase functional redundancy and is expected to stabilize ecosystem functions. Few studies, however, have explored how agricultural intensity affects functional redundancy and its link with ecosystem function stability. Here, within a continental‐wide study, we assess how functional redundancy of seed predation is affected by agricultural intensity and landscape simplification. By combining carabid abundances with molecular gut content data, functional redundancy of seed predation was quantified for 65 weed genera across 60 fields in four European countries. Across weed genera, functional redundancy was reduced with high field management intensity and simplified crop rotations. Moreover, functional redundancy increased the spatial stability of weed seed predation at the field scale. We found that ecosystem functions are vulnerable to disturbances in intensively managed agroecosystems, providing empirical evidence of the importance of biodiversity for stable ecosystem functions across space. [ABSTRACT FROM AUTHOR]

The use of sensitive methods is key for the detection of target taxa from trace amounts of environmental DNA (eDNA) in a sample. In this context, digital PCR (dPCR) enables direct quantification and is commonly perceived as more sensitive than endpoint PCR. However, endpoint PCR coupled with capillary electrophoresis (celPCR) potentially embodies a viable alternative as it quantitatively measures signal strength after PCR in Relative Fluorescence Units (RFU). Provided comparable levels of sensitivity are reached, celPCR permits the development of cost-efficient multiplex reactions, enabling the simultaneous detection of several target taxa. Here, we compared the sensitivity of singleplex and multiplex celPCR to dPCR for species-specific primer pairs amplifying mitochondrial DNA (COI) of fish species occurring in European freshwaters by analyzing dilution series of tissue extracts as well as field-collected water samples. Both singleplex and multiplex celPCR and dPCR displayed comparable sensitivity with reliable positive amplifications starting at two to 10 target DNA copies per μl extract. celPCR was suitable for quantifying target DNA and direct inference of copy numbers from RFU was possible after accounting for primer effects in linear mixed-effects models and calibration via dPCR. Furthermore, multiplex celPCR and dPCR were successfully used for the detection and quantification of fish-eDNA in field-collected water samples, confirming the results of the dilution series experiment and exemplifying the high sensitivity of the two approaches. The possibility of detection and quantification via multiplex celPCR is appealing for the cost-efficient screening of high sample numbers. The present results confirm the sensitivity of this approach thus enabling its application for future eDNA-based monitoring efforts. [ABSTRACT FROM AUTHOR]

Understanding trophic interactions in agroecosystems is crucial for harnessing ecosystem services such as pest control, thus enabling a reduction in pesticide use. Carabid beetles (Coleoptera: Carabidae) have the potential to regulate not only insect pests but also weed seeds and slugs. The aim of this study was to investigate the food choice of different carabid species in the experimental setting of a cereal field with varying seed and slug prey availability during the season. In addition to varying food availability, the effects of species identity and season on carabid food choice should also be closely examined. Therefore, the gut contents of 1,120 beetles of eight carabid species were screened for the DNA of plants, aphids, springtails, earthworms and slugs via diagnostic multiplex PCR and a nested metabarcoding approach for plant species identification. Plant DNA was detected far more often (72%) than the various animal prey types (less than 12.5% each). Within the plant detections, 80 weed species were identified in the metabarcoding, with Galinsoga parviflora/quadriradiata (Galinsoga spp.—quickweeds) as the most frequently detected species. Carabid food choice was driven by their species identity and seasonality, while no effect of increased availability of seeds and slugs on their food choice was detected. While weed seeds seem to be an important food source for carabids, their availability does not directly affect the carabid diet. The importance of consumer identity and seasonality highlight the need for a diverse carabid species community for resilient pest control services. [ABSTRACT FROM AUTHOR]

Knowledge of trophic interaction is necessary to understand the dynamics of ecosystems and develop ecosystem‐based management. The key data to measure these interactions should come from large‐scale diet analyses with good taxonomic resolution. To that end, molecular methods that analyze prey DNA from guts and feces provide high‐resolution dietary taxonomic data. However, molecular diet analysis may also produce unreliable results if the samples are contaminated by external sources of DNA. Employing the freshwater European whitefish (Coregonus lavaretus) as a tracer for sample contamination, we studied the possible route of whitefish in beaked redfish (Sebastes mentella) guts sampled in the Barents Sea. We used whitefish‐specific COI primers for diagnostic analysis, and fish‐specific 12S and metazoa‐specific COI primers for metabarcoding analyses of intestine and stomach contents of fish samples that were either not cleaned, water cleaned, or bleach cleaned after being in contact with whitefish. Both the diagnostic and COI metabarcoding revealed clear positive effects of cleaning samples as whitefish were detected in significantly higher numbers of uncleaned samples compared to water or bleach‐cleaned samples. Stomachs were more susceptible to contamination than intestines and bleach cleaning reduced the frequency of whitefish contamination. Also, the metabarcoding approach detected significantly more reads of whitefish in the stomach than in intestine samples. The diagnostic analysis and COI metabarcoding detected contaminants in a higher and comparable number of gut samples than the 12S‐based approach. Our study underlines thus the importance of surface decontamination of aquatic samples to obtain reliable diet information from molecular data. [ABSTRACT FROM AUTHOR]

The analysis of environmental DNA (eDNA) is revolutionizing the monitoring of biodiversity as it allows to assess organismic diversity at large scale and unprecedented taxonomic detail. However, eDNA consists of an extracellular and intracellular fraction, each characterized by particular properties that determine the retrievable information on when and where organisms live or have been living. Here, we review the fractions of eDNA, describe how to obtain them from environmental samples and present a four‐scenario concept that aims at enhancing spatial and temporal resolution of eDNA‐based monitoring. Importantly, we highlight how the appropriate choice of eDNA fractions precludes misinterpretation of eDNA‐based biodiversity data. Finally, future avenues of research towards eDNA fraction‐specific analyses are outlined to unravel the full potential of eDNA‐based studies targeting micro‐ and macro‐organisms. [ABSTRACT FROM AUTHOR]

Facultative scavenging by predatory carnivores is a prevalent but frequently underestimated feeding strategy. DNA‐based methods for diet analysis, however, do not allow to distinguish between scavenging and predation, thus, the significance of scavenging on population dynamics and resource partitioning is widely unknown. Here, we present a methodological innovation to differentiate between scavenging and fresh prey consumption using prey RNA as a target molecule. We hypothesized that the rapid post‐mortem breakdown of RNA in prey tissue should lead to a significantly lower detection probability of prey RNA than DNA when carrion rather than fresh prey is consumed. To test this hypothesis, ground beetles (Pseudoophonus rufipes [De Geer]) were offered either fresh or 1‐day‐old dead Drosophila melanogaster fruit flies (carrion). The detectability of prey RNA and DNA in the beetles' regurgitates was assessed with diagnostic Drosophila‐specific RT‐PCR and PCR assays at 0, 6, 12, 24 and 48 h post‐feeding. After fresh fly consumption, prey RNA and DNA were detectable equally well at all times. When carrion prey was consumed, the detection strength of prey RNA immediately after feeding was significantly lower than that of prey DNA and reached zero in most samples within 6 h of digestion. Our findings provide evidence that prey RNA allows distinguishing between the consumption of fresh and scavenged prey, thereby overcoming a long‐known weakness of molecular diet analysis. The assessment of prey RNA offers a generally applicable approach for examining the importance of scavenging in food webs to unravel its functional consequences for populations, communities, and ecosystems. [ABSTRACT FROM AUTHOR]

Piscivorous birds in aquatic ecosystems exert predation pressure on fish populations. But the site‐specific impact on fish populations, including stocked and commercially used fish species, remains disputed. One of the key questions for the management of piscivorous birds and fish is determining the origin of prey and thus which fish populations are targeted by the birds. We addressed this question by provenancing otoliths (earstones) of fish obtained from regurgitated pellets of piscivorous birds by otolith microchemistry analysis. We retrieved otoliths from regurgitated pellets of great cormorants (Phalacrocorax carbo sinensis) collected every 2 weeks for 2 years from breeding and roosting colonies at Chiemsee in Bavaria, Germany, and classified them according to family or species. We collected water samples from Chiemsee and potential surrounding foraging grounds. We measured the strontium (Sr) 87Sr/86Sr isotope ratio and Sr mass fraction of water and otoliths using (laser ablation) inductively coupled plasma‐mass spectrometry. We assigned otoliths from regurgitated pellets to habitat clusters of origin by comparing the Sr isotopic and elemental composition of otoliths and waterbodies. In 36% of cormorant pellets collected at Chiemsee, prey was assigned to waterbodies distinct from Chiemsee. Furthermore, cormorants used different foraging sites during 1 day. Microchemical provenancing of prey remains can contribute to identifying foraging sites of piscivorous birds and to what extend the birds switched among foraging sites. [ABSTRACT FROM AUTHOR]

Soil-dwelling insect pests may cause considerable damage to crops worldwide, and their belowground lifestyle makes them hard to control. Amongst the most promising control agents for subterranean pests are soilborne entomopathogenic fungi (EPF) such as Metarhizium brunneum. Albeit EPF can be highly pathogenic to their target pest species under laboratory conditions, their efficacy in the field is often limited due to adverse environmental conditions. Here, we test for the first time if the efficacy of EPF can be improved when they are augmented with trap crops. In a field experiment, the M. brunneum strain ART2825 was combined with a trap crops mixture of six plant species and evaluated for its control effect of wireworms (Coleoptera: Elateridae). When both were combined in the main crop, potato damage was lowered on average by 42.5% and wireworm abundance by 50.8%. Single application of trap crops or EPF lowered damage/pest abundance only by 29.9%/15.89% and 34.7%/4.77%, respectively. Importantly, the strength of the synergistic pest control effect between trap crops and EPF increased disproportionately with increasing wireworm abundance. However, DNA-based gut content analysis showed that wireworms’ feeding preferences were not shifting toward the trap crops. Our findings demonstrate that combining trap crops with EPF improves the efficacy of the latter and leads to a synergistic control effect which magnifies with increasing wireworm abundance. Hence, the synergistic effect of EPF and trap crops might be a promising control strategy for soil-dwelling pests in general and significantly improve our abilities to manage soil pests environmentally friendly. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "Consumer identity but not food availability affects carabid diet in cereal crops" published in the Journal of Pest Science. The correction addresses a typesetting error in Table s1 and provides the correct table and legend. The supplementary material, including a graph, is available online. The correction is authored by Yasemin Guenay-Greunke, Harald Trager, David A. Bohan, Michael Traugott, and Corinna Wallinger. Springer Nature, the publisher, remains neutral regarding jurisdictional claims and institutional affiliations. [Extracted from the article]

The focus of this study was to assess the potential of otolith microchemistry as a fishery management tool for different European freshwater fish species in an Alpine foreland with a diverse range of different water bodies but low geological variation. 87Sr/86Sr isotope ratios and Sr/Ca ratios in water samples from 26 habitat sites in a pre-alpine catchment region around lake Chiemsee, Germany, an important region for recreational and economic fisheries, were analysed. 87Sr/86Sr isotope ratios and the Sr/Ca ratios in otoliths of 246 fish out of 16 species were determined using (laser ablation) inductively coupled plasma mass spectrometry ((LA)-ICP-MS). Habitats could be differentiated into seven clusters when using the 87Sr/86Sr isotope and Sr/Ca ratios in water in combination. When using the 87Sr/86Sr isotope ratio as individual parameter, the strontium isotope groups (SIGs) based on the 87Sr/86Sr isotope ratios including the measurement uncertainties justified a three-cluster solution. The direct comparison of 87Sr/86Sr isotope ratios in water and otolith samples allowed to identify fish that a) might have migrated, b) were transferred from other water bodies or c) stocked from fish farms. Sr/Ca ratios in water and otoliths were highly correlated, although significant differences between species from the same environment were documented. Sr/Ca ratios in sagittae of Perca fluviatilis were about 64–60% of those in sagittae of Coregonus spp and of lapilli of roach Rutilus rutilus from the same habitats, respectively. Partition coefficients for water to otolith Sr/Ca ratios were calculated and differed between species. Discrimination of fish otoliths according to habitat clusters by combining 87Sr/86Sr and Sr/Ca ratios was possible with success rates of up to 100% for whitefish Coregonus spp., 90% for European perch Perca fluviatilis, 87% for cyprinids, and 89% European grayling Thymallus thymallus, and 68% for salmonids. When using the 87Sr/86Sr isotope ratios as single parameter, assignment results were generally worse, except for salmonids, where an improved accuracy of 79% could be achieved, or for cyprinids when the number of clusters was reduced from five to three. The study indicates that otolith microchemistry has great potential to serve as a fishery management tool at smaller spatial scales such as in the studied Alpine foreland when combining the 87Sr/86Sr and Sr/Ca ratios, and considering the type and spatial distribution of habitats, the species and question under investigation. • Otolith microchemistry applied in an area with limited geological variability. • Fish transferred, stocked or migrated were identified. • Sr/Ca ratios in water predict Sr/Ca ratios in otoliths. • Fish species-specific Sr discrimination from water into otoliths. • European freshwater fish species assigned to habitat clusters of origin. [ABSTRACT FROM AUTHOR]

Error: All PCR products were analysed using the QIAxcel capillary electrophoresis system (Qiagen) as described in Wallinger et al. (2015), with the exception that the method AM3201 was used. This should be changed to: The 10 µl reactions contained 2.5 µl DNA extract, 0.5 µl BSA (10 mg/ml), 5 µl 2xKAPA2G (KAPA Biosystems, Wilmington, MA, USA), 1 µl PCR-grade water and 1 µl primer mix in their respective concentrations (Table 1). 2. [Extracted from the article]