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.

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.

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.

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.

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.

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.

Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples.

Molecular techniques have become an important tool to empirically assess feeding interactions. The increased usage of next-generation sequencing approaches has stressed the need of fast DNA extraction that does not compromise DNA quality. Dietary samples here pose a particular challenge, as these demand high-quality DNA extraction procedures for obtaining the minute quantities of short-fragmented food DNA. Automatic high-throughput procedures significantly decrease time and costs and allow for standardization of extracting total DNA. However, these approaches have not yet been evaluated for dietary samples. We tested the efficiency of an automatic DNA extraction platform and a traditional CTAB protocol, employing a variety of dietary samples including invertebrate whole-body extracts as well as invertebrate and vertebrate gut content samples and feces. Extraction efficacy was quantified using the proportions of successful PCR amplifications of both total and prey DNA, and cost was estimated in terms of time and material expense. For extraction of total DNA, the automated platform performed better for both invertebrate and vertebrate samples. This was also true for prey detection in vertebrate samples. For the dietary analysis in invertebrates, there is still room for improvement when using the high-throughput system for optimal DNA yields. Overall, the automated DNA extraction system turned out as a promising alternative to labor-intensive, low-throughput manual extraction methods such as CTAB. It is opening up the opportunity for an extensive use of this cost-efficient and innovative methodology at low contamination risk also in trophic ecology.

An effective molecular approach for assessing cereal aphid-parasitoid-endosymbiont networks.

Molecular approaches are increasingly being used to analyse host-parasitoid food webs as they overcome several hurdles inherent to conventional approaches. However, such studies have focused primarily on the detection and identification of aphids and their aphidiid primary parasitoids, largely ignoring primary parasitoid-hyperparasitoid interactions or limiting these to a few common species within a small geographical area. Furthermore, the detection of bacterial secondary endosymbionts has not been considered in such assays despite the fact that endosymbionts may alter aphid-parasitoid interactions, as they can confer protection against parasitoids. Here we present a novel two-step multiplex PCR (MP-PCR) protocol to assess cereal aphid-primary parasitoid-hyperparasitoid-endosymbiont interactions. The first step of the assay allows detection of parasitoid DNA at a general level (24 primary and 16 hyperparasitoid species) as well as the species-specific detection of endosymbionts (3 species) and cereal aphids (3 species). The second step of the MP-PCR assay targets seven primary and six hyperparasitoid species that commonly occur in Central Europe. Additional parasitoid species not covered by the second-step of the assay can be identified via sequencing 16S rRNA amplicons generated in the first step of the assay. The approach presented here provides an efficient, highly sensitive, and cost-effective (~consumable costs of 1.3 € per sample) tool for assessing cereal aphid-parasitoid-endosymbiont interactions.

How generalist herbivores exploit belowground plant diversity in temperate grasslands.

Belowground herbivores impact plant performance, thereby inducing changes in plant community composition, which potentially leads to cascading effects onto higher trophic levels and ecosystem processes and productivity. Among soil-living insects, external root-chewing generalist herbivores have the strongest impact on plants. However, the lack of knowledge on their feeding behaviour under field conditions considerably hampers achieving a comprehensive understanding of how they affect plant communities. Here, we address this gap of knowledge by investigating the feeding behaviour of Agriotes click beetle larvae, which are common generalist external root-chewers in temperate grassland soils. Utilizing diagnostic multiplex PCR to assess the larval diet, we examined the seasonal patterns in feeding activity, putative preferences for specific plant taxa, and whether species identity and larval instar affect food choices of the herbivores. Contrary to our hypothesis, most of the larvae were feeding-active throughout the entire vegetation period, indicating that the grassland plants are subjected to constant belowground feeding pressure. Feeding was selective, with members of Plantaginaceae and Asteraceae being preferred; Apiaceae were avoided. Poaceae, although assumed to be most preferred, had an intermediate position. The food preferences exhibited seasonal changes, indicating a fluctuation in plant traits important for wireworm feeding choice. Species- and instar-specific differences in dietary choice of the Agriotes larvae were small, suggesting that species and larval instars occupy the same trophic niche. According to the current findings, the food choice of these larvae is primarily driven by plant identity, exhibiting seasonal changes. This needs to be considered when analysing soil herbivore-plant interactions.

Plant diversity affects behavior of generalist root herbivores, reduces crop damage, and enhances crop yield.

Soil-dwelling pests inflict considerable economic damage in agriculture but are hard to control. A promising strategy to reduce pest pressure on crops is to increase the plant diversity in agroecosystems. This approach, however, demands a sound understanding of species' interactions, which is widely lacking for subterranean herbivore-plant systems. Here, we examine the effects of plant diversification on wireworms, the soil-dwelling larvae of click beetles that threaten crops worldwide. We conducted a field experiment employing plant diversification by adding either wheat or a mix of six associated plants (grasses, legumes, and forbs) between rows of maize to protect it from Agriotes wireworms. Wireworm feeding behavior, dispersal between crop and associated plants, as well as maize damage and yield were examined. The former was assessed combining molecular gut content and stable isotope analysis. The pests were strongly attracted by the associated plants in August, when the crop was most vulnerable, whereas in September, shortly before harvest, this effect occurred only in the plant mix. In maize monoculture, the larvae stayed in the principal crop throughout the season. Larval delta13C signatures revealed that maize feeding was reduced up to sevenfold in wireworms of the vegetationally diversified treatments compared to those of the maize monoculture. These findings were confirmed by molecular analysis, which additionally showed a dietary preference of wireworms for specific plants in the associated plant mix. Compared to the monoculture, maize damage was reduced by 38% and 55% in the wheat and plant mix treatment, which translated into a yield increase of 30% and 38%, respectively. The present findings demonstrate that increasing the plant diversity in agroecosystems provides an effective insurance against soil pests. The underlying mechanisms are the diversion of the pest from the principle crop and a changed feeding behavior. The deployment of diverse mixes of associated plants, tailored to the specific preferences of the soil herbivores, provides a promising strategy for managing subterranean pests while maintaining crop yield.

Occurrence of Agriotes wireworms in Austrian agricultural land.

Agriotes wireworms (Coleoptera: Elateridae) are abundant soil-dwelling herbivores which can inflict considerable damage to field crops. In Europe up to 40 species occur, differing in their ecology and pest status. Their distribution in the larval stage, however, has rarely been assessed because of the considerable effort in collecting wireworms and the difficulties in identifying them to species-level. Here, we examined the occurrence of Agriotes wireworms in Austrian agricultural land with regard to their association with climatic and soil parameters. Using a molecular identification system, 1348 field-collected larvae from 85 sites were identified to species-level. Three species, Agriotes obscurus, Agriotes brevis, Agriotes ustulatus, and two that could not be discerned molecularly (Agriotes lineatus and Agriotes proximus), were assigned to two ecological groups: (i) A.brevis/A. ustulatus, found in areas with a warmer, drier climate and alkaline soils, and (ii) A. obscurus/A. lineatus/proximus which occur mainly at higher altitude characterised by lower temperatures, higher precipitation and acidic, humus-rich soils. Agriotes sputator was abundant throughout Austria, confirming its euryoecious nature. Only one larva of Agriotes litigiosus was found, prohibiting further analysis. These data contribute to a characterisation of species-specific traits in Agriotes larvae in agricultural land, an important prerequisite to develop efficient control strategies for these wireworms.

The effect of plant identity and the level of plant decay on molecular gut content analysis in a herbivorous soil insect.

Plant roots represent an important food source for soil-dwelling animals, but tracking herbivore food choices below-ground is difficult. Here, we present an optimized PCR assay for the detection of plant DNA in the guts of invertebrates, using general plant primers targeting the trnT-F chloroplast DNA region. Based on this assay, we assessed the influence of plant identity on the detectability of ingested plant DNA in Agriotes click beetle larvae. Six different plant species were fed to the insects, comprising a grass, a legume and four nonlegume forbs. Moreover, we examined whether it is possible to amplify DNA of decaying plants and if DNA of decayed plant food is detectable in the guts of the larvae. DNA of the ingested roots could be detected in the guts of the larvae for up to 72-h post-feeding, the maximum digestion time tested. When fed with living plants, DNA detection rates differed significantly between the plant species. This may be ascribed to differences in the amount of plant tissue consumed, root palatability, root morphology and/or secondary plant components. These findings indicate that plant identity can affect post-feeding DNA detection success, which needs to be considered for the interpretation of molecularly derived feeding rates on plants. Amplification of plant DNA from decaying plants was possible as long as any tissue could be retrieved from the soil. The consumption of decaying plant tissue could also be verified by our assay, but the insects seemed to prefer fresh roots over decaying plant material.

Rapid plant identification using species- and group-specific primers targeting chloroplast DNA.

Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae), the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory.

Effects of plant identity and diversity on the dietary choice of a soil-living insect herbivore.

Plant identity and diversity influence herbivore communities in many different ways. While it is well known how they affect the feeding preferences of aboveground herbivores, this information is lacking for soil ecosystems, where examining plant-herbivore trophic interactions is difficult. We performed a mesocosm experiment assessing how plant identity and diversity affect the food choice of Agriotes larvae, which are soil-living generalist herbivores. We offered four plant species, (maize, a grass, a legume, and a forb) at varying combinations and diversity levels to these larvae, and analyzed their feeding behavior using stable isotopes. We hypothesized that (1) their food choice is driven by preference for certain plant species rather than by root abundance and that (2) the preference for specific plants changes with increasing plant diversity. We found that larvae preferred the grass and legume but avoided maize and the forb. Whether a plant was preferred or avoided was independent of diversity, but the extent of avoidance or preference changed with increasing plant diversity. Our findings reveal that the dietary choice of soil-living generalist herbivores is determined by plant-specific traits rather than root abundance. Our data also suggest that soil herbivore feeding preferences are modulated by plant diversity.

Detecting ingested plant DNA in soil-living insect larvae.

Although a significant proportion of plant tissue is located in roots and other below-ground parts of plants, little is known on the dietary choices of root-feeding insects. This is caused by a lack of adequate methodology which would allow tracking below-ground trophic interactions between insects and plants. Here, we present a DNA-based approach to examine this relationship. Feeding experiments were established where either wheat (Triticum aestivum) or maize (Zea mays) was fed to Agriotes larvae (Coleoptera: Elateridae), allowing them to digest for up to 72 h. Due to the very small amount of plant tissue ingested (max = 6.76 mg), DNA extraction procedures and the sensitivity of polymerase chain reaction (PCR) had to be optimized. Whole-body DNA extracts of larvae were tested for the presence of both rbcL and trnL plastid DNA using universal primers. Moreover, based on cpDNA sequences encoding chloroplast tRNA for leucine (trnL), specific primers for maize and wheat were developed. With both, general and specific primers, plant DNA was detectable in the guts of Agriotes larvae for up to 72 h post-feeding, the maximum time of digestion in these experiments. No significant effect of time since feeding on plant DNA detection success was observed, except for the specific primers in maize-fed larvae. Here, plant DNA detection was negatively correlated with the duration of digestion. Both, meal size and initial mass of the individual larvae did not affect the rate of larvae testing positive for plant DNA. The outcomes of this study represent a first step towards a specific analysis of the dietary choices of soil-living herbivores to further increase our understanding of animal-plant feeding interactions in the soil.