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Abstract: To understand soil food webs, empirically generated data on the trophic connections and the feeding ecology of the major below-ground animal taxa are needed. Here we used stable isotope analysis to assess the trophic ecology of wireworms, the larvae of click beetles, in Central European arable land. Wireworms are amongst the major soil macroinvertebrates and are of practical importance in arable soils. Besides feeding on crops, they are thought to feed on weeds, soil organic matter (SOM), and even animal prey, but their feeding ecology is poorly studied under natural conditions. Elaterid larvae and their putative feeding substrates—plant roots, SOM, and litter—were sampled at 17 locations in Austria, Germany, and Italy and their isotope ratios of carbon (12C/13C) and nitrogen (14N/15N) measured to determine the wireworms’ trophic level, the importance of SOM and weeds within the diet of Agriotes larvae, as well as the individual diet variation in Agriotes obscurus larvae. δ 15N signatures suggested that Agriotes larvae are predominately herbivorous, whereas the other wireworm species primarily fed on animal prey. In contrast to SOM, weeds were readily eaten by Agriotes larvae: their dietary contribution ranged between 28% and 67% in weedy maize fields. Most A. obscurus larvae fed on a mixed diet of weeds and maize, although ∼15% of the larvae fed primarily on one of the two food sources only. δ 15N signatures indicated that ∼10% of the “herbivorous”A. obscurus larvae fed primarily on animal prey, revealing high intraspecific trophic plasticity in these soil insects. Wireworm feeding behaviour is apparently complex at the individual level: the population consists of types A and B generalists, a phenomenon which needs further assessment. [Copyright &y& Elsevier]

Abstract: Carbon and nitrogen isotope ratios in consumer tissues can be used to analyse the diet and trophic level of soil animals. However, life history traits may significantly influence stable isotope patterns. We evaluated in a series of experiments how stable isotope ratios of carbon (13C/12C) and nitrogen (15N/14N) at natural abundance can be used to study the diet and trophic position of long-lived macro-invertebrates, elaterid larvae, which are major below-ground herbivores. Small, but significant differences in δ 13C signatures were found between the larvaes’ anterior and posterior body segments, whereas exuvia reflected the body''s overall isotopic composition. The species-specific trophic shift (±SE) in δ 15N for Agriotes obscurus and Agriotes sputator (1.62±0.24‰ and 1.08±0.27‰, respectively) was significantly lower than “mean enrichment estimates” reported in the literature, showing the limited applicability of such generalised estimates in studies of invertebrate trophic ecology. To avoid false-positive assignments to two trophic levels due to variation in δ 15N values, a minimum sample size of three and five individuals for A. obscurus and A. sputator, respectively, was needed to reduce this risk to below α=5%. Keeping elaterid larvae for up to 128 days without food did not affect their isotopic signatures, in contrast to previous studies on starving animals. Switching wireworms to isotopically different diets induced changes in their isotopic signatures within 2 weeks. Changes, however, were significant only when the isotopic difference between diets was large. We conclude that experimental studies evaluating how specific life history traits affect stable isotope signatures in consumers have to precede any interpretation of stable isotope data gathered in the field. [Copyright &y& Elsevier]

Abstract: Polymerase chain reaction (PCR) can be used to detect prey within the gut contents of predators and allows specific trophic interactions to be studied among soil-dwelling invertebrates which cannot be examined by other approaches. PCR-inhibitory substances, however, are commonly found in DNA prepared from soil organisms or from biological material contaminated with soil. This can lead to false-negative results and the risk of not detecting trophic connections or of underestimating predation rates in field studies. In the present study, we developed mitochondrial DNA markers to detect Amphimallon solstitiale (Coleoptera: Scarabaeidae) in the gut contents of invertebrate predators. Larvae of A. solstitiale can cause serious damage in grasslands, field crops, and forests by feeding on roots. Adequate methodologies to study predation on these pests are lacking, and their invertebrate predator guild is, therefore, barely known. To test the new molecular markers for prey detection, larvae and eggs of A. solstitiale were fed to Poecilus versicolor larvae (Coleoptera: Carabidae), which are abundant below-ground predators in grassland ecosystems. Unfortunately, even when specific DNA extraction and purification methods were used, DNA extracts from predators were of poor quality and not amplifiable by PCR; this yielded false-negative results and a dramatically lower prey-detection rate. We overcame PCR-inhibition by applying ⩾1.28μgμl−1 bovine serum albumin to the PCR reaction mix. This enabled us to detect A. solstitiale DNA within fed carabid larvae up to 48 and 40h post-feeding for 127 and 463bp sized DNA fragments, respectively. When single A. solstitiale eggs were consumed by the carabid larvae, predation could be verified in 100% of the predators within the first 8h of digestion; some carabid larvae even tested positive 32h after feeding. Moreover, by multiplexing primers targeting both prey and predator, we were able to simultaneously screen for prey consumption and check for co-purified PCR inhibitors. Sensitivity in prey detection was not reduced compared to singleplex PCR. We recommend the multiplex approach because it considerably reduces time and costs compared to singleplex assays. We also show that multiplex PCR not only detects specific prey, but also can identify the predator itself. This allows the identification of taxa which are difficult or not identifiable based on morphological characters, such as soil-dwelling predatory beetle larvae. [Copyright &y& Elsevier]

Abstract: Earthworms play a key role in soil ecology as they can reach high densities, are well known as soil engineers, and occupy a central position in soil food-webs. The identification of earthworms, however, is notoriously difficult and morphologically only possible for well-preserved adult specimens. Molecular markers could facilitate earthworm identification and would be a huge advantage in studies where it is important to species-specifically identify juveniles or where badly preserved specimens and remains of earthworms need to be identified. The aim of this study was to compare four mitochondrial genes (12S rRNA, 16S rRNA, COI, and COII) with respect to their value for identifying earthworms, calculating earthworm phylogenies, and discovering cryptic species. Our results indicate that all four genes are suitable for species identification. However, the genetic distances were approximately twice as high for the protein coding genes than for RNA coding ones. High genetic distances and deep genetic lineages, e.g. for Octolasion lacteum, Lumbricus rubellus and Aporrectodea rosea, indicate the possible presence of cryptic species and hamper molecular identification. The Bayesian analysis based on concatenated sequence data resulted in a phylogenetic tree with high posterior probabilities. The con-specific relationship of Aporrectodea spp. and Allolobophora spp. was not confirmed, underlining the ongoing discussion about the revision of these two genera. In conclusion, our findings suggest using 12S and 16S rRNA sequences as molecular markers for species identification whereas the COI gene is better suited to address genetic lineages and to explore possible cryptic species. Taxonomy and the identification of species are essential for most ecological studies. This study provides the needed molecular sequence information to develop molecular tools that can overcome many hurdles in studies on earthworms, their relationship and their ecology. [Copyright &y& Elsevier]

Abstract: Specific primers can be used in polymerase chain reactions (PCR) to amplify prey DNA from the gut content of generalist predators at high specificity and sensitivity. A prerequisite for applying this approach to field studies, however, is to confirm that primers are actually targeting specific prey species or prey groups and do not produce false positive results by amplifying DNA either from predator species or from the wide range of potential alternative prey found under natural conditions. Here, we report on a new group-specific primer pair for earthworms designed from cytochrome oxidase c subunit 1 (COI) sequences of 11 earthworm species found in Central Europe that can be used to detect consumption of earthworms by invertebrate predators. Besides inter-specific also considerable intra-specific variation was found for COI sequences among most of the earthworm species. We, therefore, combined a universal forward primer with an earthworm-specific reverse primer which amplified a 523bp product from all 11 species tested. Earthworm DNA amplification was also successful in the presence of excess DNA of a predator species. The primer pair was tested against 82 non-target invertebrate species commonly found in the same habitats, including potential prey for generalist predators and predators themselves. The earthworm primer was highly specific: only one of the non-target species showed a product of similar length as the earthworms, whereas PCR with 12 non-target species produced amplicons whose length differed from that of earthworms. We conclude that the new primer will be a useful tool to investigate the role earthworms play as a food resource in soil food-webs. Moreover, we suggest that future studies utilizing DNA-based approaches for prey detection should select non-target species for cross-reactivity tests according to their abundance and importance rather than choosing representatives of taxonomic units; this will help validate the results achieved using species- or group-specific primers and guarantee their meaningful ecological interpretation. [Copyright &y& Elsevier]

Abstract: Soils harbour an extraordinary diversity of animals which are functionally connected by their feeding interactions. Determining these feeding links, however, is difficult due to the opaque habitat the animals live in and because many prey remains found in predator gut contents cannot be identified morphologically. DNA-based gut content analysis overcomes these hurdles. This approach opens new possibilities to empirically examine below-ground feeding links by measuring prey DNA detection frequencies in various predators. Comparing prey DNA detection rates between different predator taxa, however, is not straightforward as digestion rates and thus post-feeding prey DNA detection intervals can be affected by predator identity. In the present study we examined the effect of predator identity for a range of soil-dwelling adult and larval beetles as well as lithobiid and geophilid centipedes. These predators were fed with white grubs (scarabaeid beetle larvae) and their post-feeding prey DNA detection intervals were established for small, medium and large prey DNA fragments. In geophilids the 50% detection probability for the medium prey DNA fragment was 98 h post-feeding whereas it ranged between 22 and 34 h in beetles. Among the different beetle species, the magnitude of the differences in detection intervals was strongly affected by the size of the prey DNA fragment targeted: the intervals showed the highest similarity in the medium-sized prey DNA fragment which was most frequently detected. Predator biomass and meal size rarely affected post-feeding prey DNA detection intervals. Our findings suggest that prey DNA detection success is similar within taxonomically related species of soil-dwelling macro-invertebrates such as beetles and centipedes. Between these groups prey detection can be adjusted according to taxon-specific digestion rates for better comparisons. Moreover, the magnitude of the predator identity effect is modulated by assay sensitivity: highly sensitive assays which target small- to medium-sized prey DNA fragments are recommended as these minimize the interspecific differences in post-feeding prey DNA detection intervals. [Copyright &y& Elsevier]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: Faecal pellets of macro-decomposers form an important component of upper soil layers. It is, however, not yet specifically known which species’ faecal pellets are important in the process of humus and soil formation. Here we present a novel approach, based on the detection of short DNA fragments, to species-specifically assign faecal pellets to their detritivorous producers. Using diagnostic polymerase chain reaction (PCR), DNA of the two millipede species Cylindroiulus fulviceps and C. meinerti, abundant macro-decomposers on European alpine pastureland, could be detected in their faecal pellets. Although in both species detection was possible up to 5 days post-defecation (no longer intervals were tested), detection rates were significantly higher in C. fulviceps (81.3%) than in Cylindroiulus meinerti (60.9%) in repetitive PCRs. Employing a binomial mixture model, the probability that the producer’s DNA was present in a faecal sample which tested negative in five subsequent PCRs was estimated as 22.0% for C. fulviceps and 41.9% for C. meinerti, suggesting to adopt a flexible stopping rule for assaying this type of samples. This proof-of-concept study demonstrates that the molecular assignment of faecal pellets to their macro-invertebrate producers is possible, offering a new approach to examine the role of decomposer faecal pellets in soil formation and functioning as well as to complement molecular profiling of faunal communities. [Copyright &y& Elsevier]

Abstract: Molecular gut content analysis provides a highly specific and sensitive tool to examine the diet of soil invertebrates. Here, we present new polymerase chain reaction (PCR) assays for the detection of twelve prey taxa common in Central European forest soils. The assays target five species of collembolans as well as dipterans, gamasid and oribatid mites, lithobiid centipedes, spiders, staphylinid beetles and woodlice at the group level, amplifying 123–299 bp long DNA fragments. Cross-reactivity tests against 119 soil invertebrate taxa confirm their specificity. These new PCR assays were found to be highly sensitive, revealing the consumption of five different prey taxa in field-collected centipedes. Thus they provide a ready-to-use approach for unravelling trophic interactions among soil arthropods. [Copyright &y& Elsevier]

Abstract: We tested whether experimental summer drought affects the transfer of recently photosynthesized carbon from plants to soil mesofauna in a subalpine meadow. From day one after 13CO2 pulse-labelling of the plant canopy, roots, collembolans and mites were enriched in δ13C in control, but not in drought plots. However, as the difference in δ13C between roots and soil animals was not affected by the drought treatment, we conclude that drought affects the tight linkage between photosynthesis and soil mesofauna primarily via functional responses of plants rather than via changes in the mesofauna. [Copyright &y& Elsevier]