Functional redundancy of weed seed predation is reduced by intensified agriculture.

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.

Consumer identity but not food availability affects carabid diet in cereal crops.

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s10340-023-01620-w.

Contaminants reach everywhere: Fish dietary samples should be surface decontaminated prior to molecular diet analysis.

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.

Laboratory protocol is important to improve the correlation between target copies and metabarcoding read numbers of seed DNA in ground beetle regurgitates.

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.

Microchemical provenancing of prey remains in cormorant pellets reveals the use of diverse foraging grounds.

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.

RNA allows identifying the consumption of carrion prey.

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.

Why eDNA fractions need consideration in biomonitoring.

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.

Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology.

High-throughput sequencing platforms are increasingly being used for targeted amplicon sequencing because they enable cost-effective sequencing of large sample sets. For meaningful interpretation of targeted amplicon sequencing data and comparison between studies, it is critical that bioinformatic analyses do not introduce artefacts and rely on detailed protocols to ensure that all methods are properly performed and documented. The analysis of large sample sets and the use of predefined indexes create challenges, such as adjusting the sequencing depth across samples and taking sequencing errors or index hopping into account. However, the potential biases these factors introduce to high-throughput amplicon sequencing data sets and how they may be overcome have rarely been addressed. On the example of a nested metabarcoding analysis of 1920 carabid beetle regurgitates to assess plant feeding, we investigated: (i) the variation in sequencing depth of individually tagged samples and the effect of library preparation on the data output; (ii) the influence of sequencing errors within index regions and its consequences for demultiplexing; and (iii) the effect of index hopping. Our results demonstrate that despite library quantification, large variation in read counts and sequencing depth occurred among samples and that the sequencing error rate in bioinformatic software is essential for accurate adapter/primer trimming and demultiplexing. Moreover, setting an index hopping threshold to avoid incorrect assignment of samples is highly recommended.

Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions.

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.

Landscape complexity promotes resilience of biological pest control to climate change.

Increased climate variability as a result of anthropogenic climate change can threaten the functioning of ecosystem services. However, diverse responses to climate change among species (response diversity) can provide ecosystems with resilience to this growing threat. Measuring and managing response diversity and resilience to global change are key ecological challenges. Here, we develop a novel index of climate resilience of ecosystem services, exemplified by the thermal resilience of predator communities providing biological pest control. Field assays revealed substantial differences in the temperature-dependent activity of predator species and indices of thermal resilience varied among predator communities occupying different fields. Predator assemblages with higher thermal resilience provided more stable pest control in microcosms where the temperature was experimentally varied, confirming that the index of thermal resilience developed here is linked to predator function. Importantly, complex landscapes containing a high number of non-crop habitat patches were more likely to contain predator communities with high thermal resilience. Thus, the conservation and restoration of non-crop habitats in agricultural landscapes-practices known to strengthen natural pest suppression under current conditions-will also confer resilience in ecosystem service provisioning to climate change.

Fish as predators and prey: DNA-based assessment of their role in food webs.

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.

The resilience of weed seedbank regulation by carabid beetles, at continental scales, to alternative prey.

Carabids are generalist predators that contribute to the agricultural ecosystem service of seedbank regulation via weed seed predation. To facilitate adoption of this ecosystem services by farmers, knowledge of weed seed predation and the resilience of seedbank regulation with co-varying availability of alternative prey is crucial. Using assessments of the seedbank and predation on seed cards in 57 cereal fields across Europe, we demonstrate a regulatory effect on the soil seedbank, at a continental scale, by groups formed of omnivore, seed-eating (granivore + omnivore) and all species of carabids just prior to the crop-harvest. Regulation was associated with a positive relationship between the activity-density of carabids and seed predation, as measured on seed cards. We found that per capita seed consumption on the cards co-varied negatively with the biomass of alternative prey, i.e. Aphididae, Collembola and total alternative prey biomass. Our results underline the importance of weed seedbank regulation by carabids, across geographically significant scales, and indicate that the effectiveness of this biocontrol may depend on the availability of alternative prey that disrupt the weed seed predation.

Monitoring spawning migrations of potamodromous fish species via eDNA.

Potamodromous fish are considered important indicators of habitat connectivity in freshwater ecosystems, but they are globally threatened by anthropogenic impacts. Hence, non-invasive techniques are necessary for monitoring during spawning migrations. The use of environmental DNA (eDNA) potentially facilitates these efforts, albeit quantitative examinations of spawning migrations remain so far mostly uncharted. Here, we investigated spawning migrations of Danube bleak, Alburnus mento, and Vimba bream, Vimba vimba, and found a strong correlation between daily visual fish counts and downstream eDNA signals obtained from filtered water samples analysed with digital PCR and end-point PCR coupled with capillary electrophoresis. By accounting for daily discharge fluctuations, it was possible to predict eDNA signal strength from the number of migrating fish: first, the whole spawning reach was taken into account. Second, the model was validated using eDNA signals and fish counts obtained from the upper half of the examined river stretch. Consequently, fish counts and their day-to-day changes could be described via an eDNA-based time series model for the whole migration period. Our findings highlight the capability of eDNA beyond delivering simple presence/absence data towards efficient and informative monitoring of highly dynamic aquatic processes such as spawning migrations of potamodromous fish species.

Resilience of ecosystem processes: a new approach shows that functional redundancy of biological control services is reduced by landscape simplification.

Functional redundancy can increase the resilience of ecosystem processes by providing insurance against species loss and the effects of abundance fluctuations. However, due to the difficulty of assessing individual species' contributions and the lack of a metric allowing for a quantification of redundancy within communities, few attempts have been made to estimate redundancy for individual ecosystem processes. We present a new method linking interaction metrics with metabolic theory that allows for a quantification of redundancy at the level of ecosystem processes. Using this approach, redundancy in the predation on aphids and other prey by natural enemies across a landscape heterogeneity gradient was estimated. Functional redundancy of predators was high in heterogeneous landscapes, low in homogeneous landscapes and scaled with predator specialisation. Our approach allows quantifying functional redundancy within communities and can be used to assess the role of functional redundancy across a wide variety of ecosystem processes and environmental factors.

Molecular analysis indicates high levels of carabid weed seed consumption in cereal fields across Central Europe.

Carabid beetles are abundant in temperate agroecosystems and can play a pivotal role as biocontrol agents. While there is good knowledge regarding their effects on invertebrate pests in some systems, comparably little is known on the rate of seed feeding under field conditions. Molecular approaches are ideally suited for investigating carabid feeding interactions; to date, however, they have only been applied to animal prey. We sampled adult carabid beetles in organic cereal fields in three regions along a Central European transect. Regurgitates from populations of the three most common species, Poecilus cupreus, Pseudoophonus rufipes and Pterostichus melanarius, were screened for plant DNA, cereal aphids, collembolans and earthworms. The frequency of carabid individuals positive for plant DNA was high (> 70%) and independent of carabid species, sex, region and the time point of sampling. Detections for non-pest and pest prey were comparably lower, with 21.6% for collembolans, 18.1% for earthworms and 4.2% for aphids, respectively. Despite the prolonged detection period of plant DNA in carabid guts, as compared to animal prey, these first results suggest that weed seeds form an important part of the adult carabid diet. It would also lend support to the hypothesis that seed-feeding carabids are biocontrol agents of weeds, with effects of regulation on the weed seedbank that depend on behavioural and contextual factors including carabid species preferences for weed seed species, their life stage and tillage practices.

When to use next generation sequencing or diagnostic PCR in diet analyses.

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.

Ecosystem function in predator-prey food webs-confronting dynamic models with empirical data.

Most ecosystem functions and related services involve species interactions across trophic levels, for example, pollination and biological pest control. Despite this, our understanding of ecosystem function in multitrophic communities is poor, and research has been limited to either manipulation in small communities or statistical descriptions in larger ones. Recent advances in food web ecology may allow us to overcome the trade-off between mechanistic insight and ecological realism. Molecular tools now simplify the detection of feeding interactions, and trait-based approaches allow the application of dynamic food web models to real ecosystems. We performed the first test of an allometric food web model's ability to replicate temporally nonaggregated abundance data from the field and to provide mechanistic insight into the function of predation. We aimed to reproduce and explore the drivers of the population dynamics of the aphid herbivore Rhopalosiphum padi observed in ten Swedish barley fields. We used a dynamic food web model, taking observed interactions and abundances of predators and alternative prey as input data, allowing us to examine the role of predation in aphid population control. The inverse problem methods were used for simultaneous model fit optimization and model parameterization. The model captured >70% of the variation in aphid abundance in five of ten fields, supporting the model-embodied hypothesis that body size can be an important determinant of predation in the arthropod community. We further demonstrate how in-depth model analysis can disentangle the likely drivers of function, such as the community's abundance and trait composition. Analysing the variability in model performance revealed knowledge gaps, such as the source of episodic aphid mortality, and general method development needs that, if addressed, would further increase model success and enable stronger inference about ecosystem function. The results demonstrate that confronting dynamic food web models with abundance data from the field is a viable approach to evaluate ecological theory and to aid our understanding of function in real ecosystems. However, to realize the full potential of food web models, in ecosystem function research and beyond, trait-based parameterization must be refined and extended to include more traits than body size.

Resolving the predator first paradox: Arthropod predator food webs in pioneer sites of glacier forelands.

Primary succession on bare ground surrounded by intact ecosystems is, during its first stages, characterized by predator-dominated arthropod communities. However, little is known on what prey sustains these predators at the start of succession and which factors drive the structure of these food webs. As prey availability can be extremely patchy and episodic in pioneer stages, trophic networks might be highly variable. Moreover, the importance of allochthonous versus autochthonous food sources for these pioneer predators is mostly unknown. To answer these questions, the gut content of 1,832 arthropod predators, including four species of carabid beetles, two lycosid and several linyphiid spider species caught in early and late pioneer stages of three glacier forelands, was screened molecularly to track intraguild and extraguild trophic interactions among all major prey groups occurring in these systems. Two-thirds of the 2,310 identified food detections were collembolans and intraguild prey, while one-third were allochthonous flying insects. Predator identity and not successional stage or valley had by far the strongest impact on the trophic interaction patterns. Still, the variability of prey spectra increased significantly from early to late pioneer stage, as did the niche width of the predators. As such the structure of pioneer arthropod food webs in recently deglaciated Alpine habitats seems to be driven foremost by predator identity while site and early successional effects contribute to a lesser extent to food web variability. Our findings also suggest that in these pioneer sites, predatory arthropods depend less on allochthonous aeolian prey but are mainly sustained by prey of local production.

The effect of plant identity and mixed feeding on the detection of seed DNA in regurgitates of carabid beetles.

Carabids are abundant in temperate agroecosystems and play a pivotal role as biocontrol agents for weed seed and pest regulation. While there is good knowledge regarding their effects on invertebrate pests, direct evidence for seed predation in the field is missing. Molecular approaches are ideally suited to investigate these feeding interactions; however, the effects of an omnivorous diet, which is characteristic for many carabid species, and seed identity on the detection success of seed DNA has not yet been investigated. In a series of feeding experiments, seeds of six different Central European weed species were fed to beetles of the species Pseudoophonus rufipes, to determine post-feeding seed DNA detection rates and how these are affected by plant identity, meal size, and chemical seed composition. Moreover, we investigated the effect of a mixed diet of seeds and mealworm on prey DNA detection. Four out of six seed species were detectable for up to five days after consumption, and seed species identity significantly affected post-feeding detection rates. Detectability was negatively influenced by protein content and seed mass, whereas oil content and meal size had a positive effect. The mixed diet led to both increased detection rates and post-feeding detection intervals of seed DNA. This suggests that mixed feeding leads to an enhancement of food detection intervals in carabid beetles and that seed identity, their chemical composition, and meal size can affect DNA detection of consumed seeds. These aspects and potential implications of this non-invasive approach are discussed as they can become highly relevant for interpreting field-derived data.