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The larvae of many hoverflies (Diptera: Syrphidae) are important polyphagous predators used in integrated pest management programs. Because the accurate identification of preimaginal stages by morphological characters is difficult, we have developed a multiplex PCR to identify the immature and/or adult stages of the most common syrphid species in Mediterranean vegetable crops: Episyrphus balteatus, Scaeva pyrastri, Eupeodes corollae, Meliscaeva auricollis, Sphaerophoria scripta, and Sphaerophoria rueppellii. The latter two species were amplified by the same primer pair due to the high similarity of their cytochrome oxidase subunit I sequences. Additionally, the assay included a primer pair targeting Diplazon laetatorius, a common koinobiont ichneumonid endoparasitoid of predatory syrphid larvae. The multiplex PCR assay proved to be highly specific and sensitive, and it was used to study the assemblage of hoverfly species in larval stage in two Mediterranean lettuce crops in two consecutive years. The molecular analysis revealed that Eu. corollae, Ep. balteatus, and Sph. scripta/Sph. rueppellii were the species present in the investigated fields. Species composition differed depending on sampling date and whether the larvae were collected on the plants or on the ground. The parasitoid D. laetatorius was not detected in any of the analyzed hoverfly larvae, suggesting low-parasitism pressure in the studied syrphid populations. The wide distribution of most of these syrphid species makes this multiplex PCR assay an ideal tool to deepen our knowledge on the ecology of these polyphagous hoverfly species in preimaginal stages and to improve the use of hoverflies to control insect pests. [ABSTRACT FROM AUTHOR]

Abstract: Food webs in agricultural systems are complex and trophic linkages are difficult to track using conventional methodologies. Here, we review three alternative approaches that allow empirical assessment of feeding interactions: DNA-based techniques, and stable isotope and fatty acid analyses. DNA-based methods, namely multiplex PCR and next-generation sequencing, allow identification of food types and host–parasitoid linkages, resulting in taxonomically highly resolved feeding networks. Stable isotopes and fatty acids reflect the assimilation of broader categories of resources, as metabolised into the consumers’ tissue, together with the associated energy and nutrient fluxes in the food web. We discuss the strengths of the approaches but also highlight their limitations, providing practical advice on which technique is best suited to answer specific questions in examining food web interactions in agroecosystems. Future refinements of these techniques, especially when used in combination, could herald a new era in agricultural food web ecology, enabling management and environmental impact to be placed in the mechanistic context of trophic networks. [Copyright &y& Elsevier]

Generalist predators such as carabid and cantharid beetles form an important component within natural enemy communities in arable land. Usually, predator-prey interactions are examined during the vegetation period, largely ignoring food web interactions occurring in the cold season. This is, however, when the larval stages of many polyphagous beetles develop whose survival critically depends on the availability of suitable prey. In this study, we examined intra- and extraguild feeding links in larval Cantharis spp. (Coleoptera: Cantharidae) and Nebria brevicollis (Coleoptera: Carabidae), both abundant cold-adapted invertebrate predators in European arable land. As these immature beetles are fluid feeders, which impedes a microscopic analysis of their gut content, multiplex polymerase chain reaction assays were developed to examine trophic linkages in the field. Collembolan DNA was detected in 49% and 54% of cantharid and carabid larvae, respectively. Earthworms were consumed by 34% of cantharid and 24% of carabid larvae. Significant differences in lumbricid and collembolan prey detection rates occurred between sampling dates in Cantharis spp. and N. brevicollis larvae, respectively, suggesting that these predators utilized different feeding strategies. In both predator taxa, however, only few individuals (0·2-1%) tested positive for DNA of intraguild prey, indicating that predator-predator trophic interactions were scarce in this community. Synthesis and applications. We present conclusive evidence that cold-adapted predatory beetle larvae are strongly linked to the detrital food chain by feeding on collembolans and earthworms. By improving the habitat conditions for detritivores in arable land by mulching, compost applications, or provision of plant cover during winter, their densities can be increased easily. Applying these measures year round will retain and sustain predatory beetles during their whole life cycle in arable land. Ultimately, we expect that these measures will enhance the ability of polyphagous beetle predators to provide their fundamental ecosystem service as regulators of agricultural pests. [ABSTRACT FROM AUTHOR]

Abstract: The use of diagnostic polymerase chain reaction (PCR) is a valuable approach to study host–parasitoid interactions. It circumvents problems inherent to rearing parasitoids from field-collected hosts or host dissection. Here, we present a PCR-based detection method for Diadegma semiclausum, Cotesia glomerata, and Cotesia rubecula, which are key-parasitoids of the three lepidopteran cabbage pests Plutella xylostella, Pieris brassicae, and Pieris rapae, respectively. Primer pairs for the three parasitoid species and Pl. xylostella were developed: they were used either separately in singleplex PCR or combined in multiplex PCR to (1) screen simultaneously for the two Cotesia species or to (2) detect the parasitoid D. semiclausum and identify its host Pl. xylostella in one PCR. The new idea to simultaneously identify parasitoid and host by molecular markers is useful when the host, in our case early-instar larvae of Pl. xylostella, is morphologically difficult to distinguish from other host species also occurring in the same habitat. Concentration–response trials revealed comparable detection sensitivity of singleplex and multiplex PCR, with detection limits ranging from 0.03 to 2.2pg of parasitoid DNA/μl PCR. Furthermore, the different developmental time of immature D. semiclausum and C. glomerata did not influence parasitoid detection success in either assay type. Based on multiplex PCR screening of field-collected caterpillars, we found in Pl. xylostella, P. brassicae, and P. rapae parasitism rates of 33.4% by D. semiclausum, 52% by C. glomerata, and 53.4% by C. rubecula, respectively. [Copyright &y& Elsevier]

White grubs (larvae of Coleoptera: Scarabaeidae) are abundant in below-ground systems and can cause considerable damage to a wide variety of crops by feeding on roots. White grub populations may be controlled by natural enemies, but the predator guild of the European species is barely known. Trophic interactions within soil food webs are difficult to study with conventional methods. Therefore, a polymerase chain reaction (PCR)-based approach was developed to investigate, for the first time, a soil insect predator-prey system. Can, however, highly sensitive detection methods identify carrion prey in predators, as has been shown for fresh prey? FreshMelolontha melolontha(L.) larvae and 1- to 9-day-old carcasses were presented toPoecilus versicolorSturm larvae. Mitochondrial cytochrome oxidase subunit I fragments of the prey, 175, 327 and 387 bp long, were detectable in 50% of the predators 32 h after feeding. Detectability decreased to 18% when a 585 bp sequence was amplified. Meal size and digestion capacity of individual predators had no influence on prey detection. Although prey consumption was negatively correlated with cadaver age, carrion prey could be detected by PCR as efficiently as fresh prey irrespective of carrion age. This is the first proof that PCR-based techniques are highly efficient and sensitive, both in fresh and carrion prey detection. Thus, if active predation has to be distinguished from scavenging, then additional approaches are needed to interpret the picture of prey choice derived by highly sensitive detection methods. [ABSTRACT FROM AUTHOR]

DNA-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. [ABSTRACT FROM AUTHOR]

The use of sensitive methods is key for the detection of target taxa from trace amounts of environmental DNA (eDNA) in a sample. In this context, digital PCR (dPCR) enables direct quantification and is commonly perceived as more sensitive than endpoint PCR. However, endpoint PCR coupled with capillary electrophoresis (celPCR) potentially embodies a viable alternative as it quantitatively measures signal strength after PCR in Relative Fluorescence Units (RFU). Provided comparable levels of sensitivity are reached, celPCR permits the development of cost-efficient multiplex reactions, enabling the simultaneous detection of several target taxa. Here, we compared the sensitivity of singleplex and multiplex celPCR to dPCR for species-specific primer pairs amplifying mitochondrial DNA (COI) of fish species occurring in European freshwaters by analyzing dilution series of tissue extracts as well as field-collected water samples. Both singleplex and multiplex celPCR and dPCR displayed comparable sensitivity with reliable positive amplifications starting at two to 10 target DNA copies per μl extract. celPCR was suitable for quantifying target DNA and direct inference of copy numbers from RFU was possible after accounting for primer effects in linear mixed-effects models and calibration via dPCR. Furthermore, multiplex celPCR and dPCR were successfully used for the detection and quantification of fish-eDNA in field-collected water samples, confirming the results of the dilution series experiment and exemplifying the high sensitivity of the two approaches. The possibility of detection and quantification via multiplex celPCR is appealing for the cost-efficient screening of high sample numbers. The present results confirm the sensitivity of this approach thus enabling its application for future eDNA-based monitoring efforts. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aphids are major pests of cereal crops and a suite of hymenopteran primary parasitoids play an important role in regulating their populations. However, hyperparasitoids may disrupt the biocontrol services provided by primary parasitoids. As such, understanding cereal aphid-primary parasitoid-hyperparasitoid interactions is vital for a reliable parasitoid-based control of cereal aphids. For this, the ability to identify the different primary and hyperparasitoid species is necessary. Unfortunately, this is often difficult due to a lack of morphologically diagnostic features. DNA sequence-based species identification of parasitoids can overcome these hurdles. However, comprehensive DNA sequence information is lacking for many of these groups, particularly for hyperparasitoids. Here we evaluate three genes [cytochrome c oxidase subunit I (COI), 16S ribosomal RNA (16S) and 18S ribosomal RNA (18S)] for their suitability to identify 24 species of primary parasitoids and 16 species of hyperparasitoids associated with European cereal aphids. To identify aphelinid primary parasitoid species and hyperparasitoids, we found 16S to be more suitable compared to COI sequences. In contrast, the Aphidiinae are best identified using COI due to better species-level resolution and a more comprehensive DNA sequence database compared to 16S. The 18S gene was better suited for group-specific identification of parasitoids, but did not provide resolution at the species level. Our results provide a DNA sequence database for cereal aphid primary parasitoids and their associated hyperparasitoids in Central Europe, which will allow further improvement of our understanding of cereal aphid-primary parasitoid-hyperparasitoid interactions in relation to aphid biological control. [ABSTRACT FROM AUTHOR]

The applicability of species-specific primers to study feeding interactions is restricted to those ecosystems where the targeted prey species occur. Therefore, group-specific primer pairs, targeting higher taxonomic levels, are often desired to investigate interactions in a range of habitats that do not share the same species but the same groups of prey. Such primers are also valuable to study the diet of generalist predators when next generation sequencing approaches cannot be applied beneficially. Moreover, due to the large range of prey consumed by generalists, it is impossible to investigate the breadth of their diet with species-specific primers, even if multiplexing them. However, only few group-specific primers are available to date and important groups of prey such as flying insects have rarely been targeted. Our aim was to fill this gap and develop group-specific primers suitable to detect and identify the DNA of common taxa of flying insects. The primers were combined in two multiplex PCR systems, which allow a time- and cost-effective screening of samples for DNA of the dipteran subsection Calyptratae (including Anthomyiidae, Calliphoridae, Muscidae), other common dipteran families (Phoridae, Syrphidae, Bibionidae, Chironomidae, Sciaridae, Tipulidae), three orders of flying insects (Hymenoptera, Lepidoptera, Plecoptera) and coniferous aphids within the genus Cinara. The two PCR assays were highly specific and sensitive and their suitability to detect prey was confirmed by testing field-collected dietary samples from arthropods and vertebrates. The PCR assays presented here allow targeting prey at higher taxonomic levels such as family or order and therefore improve our ability to assess (trophic) interactions with flying insects in terrestrial and aquatic habitats. [ABSTRACT FROM AUTHOR]

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 trn T- 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. [ABSTRACT FROM AUTHOR]

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 semidigested 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. [ABSTRACT FROM AUTHOR]

Molecular methods have become an important tool for studying feeding interactions under natural conditions. Despite their growing importance, many methodological aspects have not yet been evaluated but need to be considered to fully exploit the potential of this approach. Using feeding experiments with high alpine carabid beetles and lycosid spiders, we investigated how PCR annealing temperature affects prey DNA detection success and how post-PCR visualization methods differ in their sensitivity. Moreover, the replicability of prey DNA detection among individual PCR assays was tested using beetles and spiders that had digested their prey for extended times postfeeding. By screening all predators for three differently sized prey DNA fragments (range 116-612 bp), we found that only in the longest PCR product, a marked decrease in prey detection success occurred. Lowering maximum annealing temperatures by 4 °C resulted in significantly increased prey DNA detection rates in both predator taxa. Among the three post-PCR visualization methods, an eightfold difference in sensitivity was observed. Repeated screening of predators increased the total number of samples scoring positive, although the proportion of samples testing positive did not vary significantly between different PCRs. The present findings demonstrate that assay sensitivity, in combination with other methodological factors, plays a crucial role to obtain robust trophic interaction data. Future work employing molecular prey detection should thus consider and minimize the methodologically induced variation that would also allow for better cross-study comparisons. [ABSTRACT FROM AUTHOR]

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: Organic farmers cannot use most commonly available synthetic insecticides to control crop pests and reduce economic losses due to pest outbreaks. Instead, they rely heavily on the activities of naturally occurring predators and parasitoids, particularly in broad-acre crops such as cereals. It is still unclear whether organic farms which typically support greater levels of biodiversity also experience greater levels of pest control services. We assess whether organic farming systems were better able to control cereal aphids due to a greater diversity and activity of naturally occurring parasitoid species. We anticipated greater parasitism rate of aphids in organic arable fields due to closer proximity to plants that provide resources for adult parasitoids and places that are suitable for overwintering, and the presence of alternate aphid hosts when cereals are absent. Aphids were collected from organic and conventional cereal crops, and screened for parasitoid DNA using diagnostic polymerase chain reaction (PCR). Aphid abundance was low across the season, however organic cereal fields had significantly greater aphid abundance. From the 1446 aphids screened we detected 12 parasitoid taxa. There was no difference in parasitism rate between the farming systems (org. mean mortality 20.9±3.3% s.e., con. 29.8±4.9%). Furthermore, there was no difference between farming systems in hyperparasitoid attack and multiparasitism rates, parasitoid richness and parasitoid community diversity. The most abundant species recorded on organic and conventional farms was the generalist aphid primary parasitoid Ephedrus plagiator. It appears that the extra plant diversity and greater area of semi-natural habitats on organic farms offer no advantage for increasing cereal aphid parasitoid diversity, at least at the aphid abundances encountered during the sampling period. [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]