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

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

A multiplex PCR assay for detecting slug species common in European arable land in the diet of carabid beetles

International audienceDNA-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

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

International audienceAbstract 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

Data from: 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 seed 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

Granivorous carabids Dryad

Table1 for the feeding experiments of Pseudoophonus rufipes fed with seeds of 5 different plant species and Table2: mixed feeding experiment of P.rufipes with Tenebrio molitor and seeds of two different plant species - detection of the food types using general molecular marker

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

International audienceCarabid 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

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

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The amount of environmental DNA increases with freshwater crayfish density and over time

eDNA analysis is ideally suited to monitor the occurrence of endangered or invasive species because of its non-invasive nature and high sensitivity. European freshwater crayfish are threatened across the whole continent. Classical crayfish monitoring is challenging and time consuming due to their nocturnal activity and hidden lifestyle. Therefore, eDNA-based monitoring of native as well as invasive species seems to be of great benefit for the conservation of the native species and it has indeed been increasingly applied in recent years. Nevertheless, comparably little is known on the relationship between eDNA concentration and crayfish population densities, a prerequisite for estimating population size based on eDNA measurements. Here, we performed laboratory experiments to investigate the relationship between the concentration of crayfish eDNA and population densities - measured as crayfish size and biomass. There was a strong correlation between the two measurements. Moreover, the amount of eDNA increased at least during the first three days after crayfish stocking in the aquarium. The experiments also indicate, that crayfish activity might have a strong influence on the eDNA signal strength. Our findings will significantly contribute to an optimization of the monitoring of freshwater crayfish via the analysis of eDNA and therefore be important for the conservation of these threatened species

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

International audienceAbstract 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