Karin Staudacher

PCR-based species identification of Agriotes larvae

Click beetle larvae within the genus Agriotes (Coleoptera: Elateridae), commonly known as wireworms, are abundant ground-dwelling herbivores which can inflict considerable damage to field crops. In Central Europe up to 20 species, which differ in their distribution, ecology and pest status, occur in arable land. However, the identification of these larvae based on morphological characters is difficult or impossible. This hampers progress towards controlling these pests. Here, we present a polymerase chain reaction (PCR)-based approach to identify, for the first time, 17 Agriotes species typically found in Central Europe. Diagnostic sequence information was generated and submitted to GenBank, allowing the identification of these species via DNA barcoding. Moreover, multiplex PCR assays were developed to identify the nine most abundant species rapidly within a single-step reaction: Agriotes brevis, A. litigiosus, A. obscurus, A. rufipalpis, A. sordidus, A. sputator, A. ustulatus, A. lineatus and A. proximus. The latter two species remain molecularly indistinguishable, questioning their species status. The multiplex PCR assays proved to be highly specific against non-agrioted elaterid beetles and other non-target soil invertebrates. By testing the molecular identification system with over 900 field-collected larvae, our protocol proved to be a reliable, cheap and quick method to routinely identify Central European Agriotes species.

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.

Diet of generalist predators reflects effects of cropping period and farming system on extra‐ and intraguild prey

The suppression of agricultural pests by natural enemies, including generalist arthropod predators, is an economically important regulating ecosystem service. Besides pests, generalist predators may also consume non-pest extraguild and intraguild prey, which can affect their impact on pest populations. This may either reduce the impact of generalist predators on pest populations, because they are diverted from pest predation, or increase it, as it helps them survive periods of low pest availability. However, the availability of pest prey and alternative, non-pest prey can vary over the crop growing season and between farming systems, potentially affecting predator-prey interactions and the levels of biological control. We have limited information about how farming systems and environmental variation over the crop growing season influence predator diets. This limits our ability to predict the importance of generalist predators as natural enemies of agricultural pests. Here we utilize molecular gut content analyses to assess detection frequencies of extra- and intraguild prey DNA in generalist predator communities in replicated organically and conventionally managed cereal fields at two key periods of the cropping season for aphid biological control. This is done in order to understand how farming system, crop season, prey availability and predator community composition determine the composition of predator diets. Aphid pests and decomposers (springtails) were equally important prey for generalist predators early in the growing season. Later in the season, the importance of aphid prey increased with increasing aphid densities while springtail predation rates were positively correlated to abundance of this prey at both early and late crop growth stages. Intraguild predation was unidirectional: carabids fed on spiders, whereas spiders rarely fed on carabids. Carabids had higher detection frequencies for the two most common spider families in organically compared to conventionally managed fields. Our study documents that predation by generalist predator communities on aphid pests increases with pest numbers independently of their generally widespread consumption of alternative, non-pest prey. Therefore, conservation strategies in agricultural fields could promote biological control services by promoting high levels of alternative non-pest prey for generalist predator communities.

Additive effects of predator diversity on pest control caused by few interactions among predator species

1. Studies of the impact of predator diversity on biological pest control have shown idiosyncratic results. This is often assumed to be as a result of differences among systems in the importance of predator–predator interactions such as facilitation and intraguild predation. The frequency of such interactions may be altered by prey availability and structural complexity. A direct assessment of interactions among predators is needed for a better understanding of the mechanisms affecting prey abundance by complex predator communities.

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

High Redundancy as well as Complementary Prey Choice Characterize Generalist Predator Food Webs in Agroecosystems

Food web structure influences ecosystem functioning and the strength and stability of associated ecosystem services. With their broad diet, generalist predators represent key nodes in the structure of many food webs and they contribute substantially to ecosystem services such as biological pest control. However, until recently it has been difficult to empirically assess food web structure with generalist predators. We utilized DNA-based molecular gut-content analyses to assess the prey use of a set of generalist invertebrate predator species common in temperate agricultural fields. We investigated the degree of specialization of predator-prey food webs at two key stages of the cropping season and analysed the link temperature of different trophic links, to identify non-random predation. We found a low level of specialization in our food webs, and identified warm and cool links which may result from active prey choice or avoidance. We also found a within-season variation in interaction strength between predators and aphid pests which differed among predator species. Our results show a high time-specific functional redundancy of the predator community, but also suggest temporally complementary prey choice due to within-season succession of some predator species.