Corinna Wallinger

Dispersal abilities of adult click beetles in arable land revealed by analysis of carbon stable isotopes

1 The dispersal abilities of agrioted beetles, serious pests on a variety of crops, are poorly known under natural conditions. This hampers their control. We used, for the first time, a stable isotope approach to assess dispersal of adult Agriotes obscurus in arable land.

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.

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.

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.

Detection of seed DNA in regurgitates of granivorous carabid beetles.

Granivory can play a pivotal role in influencing regeneration, colonization as well as abundance and distribution of plants. Due to their high abundance, nutrient content and longevity, seeds are an important food source for many animals. Among insects, carabid beetles consume substantial numbers of seeds and are thought to be responsible for a significant amount of seed loss. However, the processes that govern which seeds are eaten and are therefore prevented from entering the seedbank are poorly understood. Here, we assess if DNA-based diet analysis allows tracking the consumption of seeds by carabids. Adult individuals of Harpalus rufipes were fed with seeds of Taraxacum officinale and Lolium perenne allowing them to digest for up to 3 days. Regurgitates were tested for the DNA of ingested seeds at eight different time points post-feeding using general and species-specific plant primers. The detection of seed DNA decreased with digestion time for both seed species, albeit in a species-specific manner. Significant differences in overall DNA detection rates were found with the general plant primers but not with the species-specific primers. This can have implications for the interpretation of trophic data derived from next-generation sequencing, which is based on the application of general primers. Our findings demonstrate that seed predation by carabids can be tracked, molecularly, on a species-specific level, providing a new way to unravel the mechanisms underlying in-field diet choice in granivores.

Understanding the ecology of wireworms and improving their control: a special issue

Wireworms, the larvae of click beetles (Coleoptera: Elateridae), are an important group of soil dwelling pests which attack the subterranean parts of a wide range of crop plants. They are found in arable soils all over the world and comprise many species. For example, in the Holarctic there are more than 100 species of economic importance to arable crops (Vernon and van Herk 2013). Aside from directly damaging or killing the crop plants, wireworm damage also often paves the way for other plant pathogens such as fungi which indirectly harm the crop. Both direct and indirect damage can inflict considerable price reductions for crops such as potatoes (Keiser et al. 2012). Currently, there are thousands of hectares of cropland which are threatened by these pests, hence effective control measures need to be brought in place. However, the control of wireworms is challenging: the opaque environment, the patchy distribution of the larvae and their seasonal movements in the soil column make it hard to monitor wireworm populations and the damage level is, therefore, difficult to predict. New monitoring approaches, including the assessment of adult populations using pheromones, are thus needed. The identification of wireworms to species level is a prerequisite to develop successful control measures, but it is challenging and represents an area where new techniques are needed to allow for reliable and rapid taxonomic assignment of larvae (Staudacher et al. 2011). The search for effective biological control agents such as entomopathogenic fungi is another important aspect to develop new measures for wireworm regulation. Aside from natural enemies, resistant crop varieties or within field measures such as trap crops (Vernon et al. 2000) have shown their potential to reduce wireworm damage. Moreover, the worldwide demand to significantly reduce the use of pesticides calls for new tactics to control wireworms using a minimum of insecticides, e.g. attract and kill approaches which also include a thorough assessment of wireworm behaviour. Finally, wireworm control does not stop at the field margin but needs to consider the spatial ecology of the pest and the impact of the landscape and climate to successfully manage agrobiont click beetle populations (Benefer et al. 2012). This special issue on the ecology and control of wireworms addresses all of the topics mentioned above. It is the product of a wireworm symposium held within the 13th European meeting of the IOBC/WPRS working group ‘Insect Pathogens and Insect Parasitic Nematodes’, in Innsbruck Austria from June 23–24, 2011. We wish to thank the participants of this inspiring symposium where the latest advances in click beetle research were presented and discussed. Particularly, we are grateful to those who provided us with papers for this special issue. This issue also links up with the long standing tradition of Journal of Pest Science in publishing research on wireworm control: in fact the very first paper published within this journal, the formerly named ‘Anzeiger fur Schaedlingskunde’, dealt with the risk of introducing wireworms into arable fields by forest litter (Escherich 1925). Now, 88 years later, we re-connect with this early work by presenting a whole issue dedicated to wireworm ecology and control. We hope that the knowledge A contribution to the Special Issue on Ecology and Control of Wireworms

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.

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

Abstract 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.