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Introgressive hybridization results in mito-nuclear discordance which could obscure the delimitation of closely related taxa. Although such events are increasingly reported, they have been poorly studied in earthworms. Here, we propose a method for investigating the degree of introgressive hybridization between three taxa of the Allolobophora chlorotica aggregate within two field populations (N = 67 and N = 105) using a reference data set including published DNA barcoding and microsatellite data of all known A. chlorotica lineages (N = 85). For this, we used both molecular phylogenetic and population genetic approaches. The test of correspondence between mitochondrial cytochrome c oxidase I (COI) lineages and clusters of nuclear microsatellite genotypes allowed individuals to be sorted in three categories (matching, admixed and nonmatching) and additional markers (mitochondrial NADH dehydrogenase subunit 1, nuclear Histone 3 and Internal transcribed Spacer Region 2) were used for phylogenetic reconstructions in order to check assignments. Although 15 admixed individuals were observed, no early-generation hybrids were detected within the two populations. Interestingly, 14 nonmatching individuals (i.e. with a mtDNA haplotype that did not correspond to their nuclear cluster) were detected, a pattern that would result after multiple generations of unidirectional hybridization of female from one taxon to male of the other taxon. Because earthworms are simultaneous hermaphrodites, these events of unidirectional hybridization suggest sterility of the male function in several crosses and highlight that some individuals can be misidentified if reliance is placed on COI barcodes alone. These findings could improve the use of these barcodes in earthworms for species delineation., (© 2016 John Wiley & Sons Ltd.)

Although agriculture is amongst the world's most widespread land uses, studies of its effects on stream ecosystems are often limited in spatial extent. National monitoring data could extend spatial coverage and increase statistical power, but present analytical challenges where covarying environmental variables confound relationships of interest.Propensity modelling is used widely outside ecology to control for confounding variables in observational data. Here, monitoring data from over 3000 English and Welsh river reaches are used to assess the effects of intensive agricultural land cover (arable and pastoral) on stream habitat, water chemistry and invertebrates, using propensity scores to control for potential confounding factors (e.g. climate, geology). Propensity scoring effectively reduced the collinearity between land cover and potential confounding variables, reducing the potential for covariate bias in estimated treatment-response relationships compared to conventional multiple regression.Macroinvertebrate richness was significantly greater at sites with a higher proportion of improved pasture in their catchment or riparian zone, with these effects probably mediated by increased algal production from mild nutrient enrichment. In contrast, macroinvertebrate richness did not change with arable land cover, although sensitive species representation was lower under higher proportions of arable land cover, probably due to greatly elevated nutrient concentrations. Synthesis and applications . Propensity modelling has great potential to address questions about pressures on ecosystems and organisms at the large spatial extents relevant to land-use policy, where experimental approaches are not feasible and broad environmental changes often covary. Applied to the effects of agricultural land cover on stream systems, this approach identified reduced nutrient loading from arable farms as a priority for land management. On this specific issue, our data and analysis support the use of riparian or catchment-scale measures to reduce nutrient delivery to sensitive water bodies.

True predators are characterised by capturing a number of prey items during their lifetime and by being generalists. Some true predators are facultative specialists, but very few species are stenophagous specialists that catch only a few closely related prey types. A monophagous true predator that would exploit a single prey species has not been discovered yet. Representatives of the spider family Ammoxenidae have been reported to have evolved to only catch termites. Here we tested the hypothesis that Ammoxenus amphalodes is a monophagous termite-eater capturing only Hodotermes mossambicus. We studied the trophic niche of A. amphalodes by means of molecular analysis of the gut contents using Next Generation Sequencing. We investigated their willingness to accept alternative prey and observed their specific predatory behaviour and prey capture efficiency. We found all of the 1.4 million sequences were H. mossambicus. In the laboratory A. amphalodes did not accept any other prey, including other termite species. The spiders attacked the lateral side of the thorax of termites and immobilised them within 1 min. The paralysis efficiency was independent of predator:prey size ratio. The results strongly indicate that A. amphalodes is a monophagous prey specialist, specifically adapted to feed on H. mossambicus.

Wild habitats adjoining farmland are potentially valuable sources of natural enemies, but also of pests. Here we tested the utility of birds as 'sampling devices', to identify the diversity of prey available to predators and particularly to screen for pests and natural enemies using natural ecosystems as refugia. Here we used PCR to amplify prey DNA from three sympatric songbirds foraging on small invertebrates in Phragmites reedbed ecosystems, namely the Reed Warbler (Acrocephalus scirpaceus), Sedge Warbler (Acrocephalus schoenobaenus) and Cetti's Warbler (Cettia cetti). A recently described general invertebrate primer pair was used for the first time to analyse diets. Amplicons were cloned and sequenced, then identified by reference to the Barcoding of Life Database and to our own sequences obtained from fresh invertebrates. Forty-five distinct prey DNA sequences were obtained from 11 faecal samples, of which 39 could be identified to species or genus. Targeting three warbler species ensured that species-specific differences in prey choice broadened the range of prey taken. Amongst the prey found in reedbeds were major pests (including the tomato moth Lacanobia oleracea) as well as many potentially valuable natural enemies including aphidophagous hoverflies and braconid wasps. Given the mobility of birds, this approach provides a practical way of sampling a whole habitat at once, providing growers with information on possible invasion by locally resident pests and the colonization potential of natural enemies from local natural habitats.

Generalist species with numerous food web interactions are thought to provide stability to ecosystem dynamics; however, it is not always clear whether habitat generality translates into dietary diversity. Big brown bats are common across North America and employ a flexible foraging strategy over water, dense forests, forest edges and rural and urban settings. Despite this generalist use of habitat, they are paradoxically characterized as beetle specialists. However, hard carapaces may preferentially survive digestion leading to over-representation during morphological analysis of diet. This specialization has not been evaluated independently using molecular analysis and species-level identification of prey. We used next-generation sequencing to assess the diet of big brown bats. Beetles were consumed in the highest frequency but Lepidoptera species richness was highest among identified prey. The consumption of species showed strong seasonal and annual variation. While Coleoptera consumption varied, Lepidoptera and Ephemeroptera were relatively constant dietary components. Dietary diversity increased in late summer when insect diversity decreases. Our results indicate that big brown bats are dietary generalists and, while beetles are an important component of the diet, Lepidoptera are equally important, and Lepidoptera and Ephemeroptera are the only stable prey resource exploited. As resources become limited, big brown bats may respond by increasing the species richness of prey and thus their connectedness in the ecosystem. This characterization of diet corresponds well with a generalist approach to foraging, making them an important species in encouraging and maintaining ecosystem stability.

Prevalence and intensity of parasitic infections are often higher in male than in female vertebrates. This bias may represent either differences between host sex in exposure or susceptibility to parasites. The former may be due to sex-specific behaviour of the host, including differential habitat use or diet. Differences in susceptibility are often regarded as a negative effect of male sex steroid hormones on the immune system. Host-parasite dynamics are of great interest in terms of reptile survival, ecology and conservation. We used, for the first time, molecular diagnostics to track nematode parasitism in wild populations of reptiles noninvasively. Using slow worms (Anguis fragilis) as a model species, we investigated the interacting effects of time of year, sex, length, weight and climatic variables on the prevalence of the gastroenterological parasitic nematode Neoxysomatium brevicaudatum. Faeces were collected from three sites over 2 years. There was an interaction between sex and time of year, with lower nematode prevalence in males than in females in July or August (different between years) but a high prevalence in males in April. As the latter is during the slow worm breeding season, this may be the result of testosterone-induced immunosuppression. A second-order interaction between slow worm length and weight was found to be significant, with a positive association between prevalence and body condition in young slow worms and a negative association in older slow worms. The convex pattern of nematode prevalence with age that emerged suggests an increase with age-related exposure and a decrease with age-related acquired immunity., (© 2014 John Wiley & Sons Ltd.)

Reptiles are declining in many parts of the world, mainly due to habitat loss and environmental change. A major factor in this is availability of suitable food. For many animals, dietary requirements shift during developmental stages and a habitat will only be suitable for conserving a species if it supports all stages. Conventional methods for establishing diet often rely on visual recognition of morphologically identifiable features of prey in faeces, regurgitation or stomach contents, which suffer from biases and poor resolution of taxa. DNA-based techniques facilitate noninvasive analysis of diet from faeces without these constraints. We tested the hypothesis that diet changes during growth stages of smooth snakes (Coronella austriaca), which have a highly restricted distribution in the UK but are widespread in continental Europe. Small numbers of the sympatric grass snake (Natrix natrix) were analysed for comparison. Faecal samples were collected from snakes and prey DNA analysed using PCR, targeting amphibians, reptiles, mammals and invertebrates. Over 85% of smooth snakes were found to have eaten reptiles and 28% had eaten mammals. Predation on mammals increased with age and was entirely absent among juveniles and subadults. Predation on reptiles did not change ontogenetically. Smooth snakes may, therefore, be restricted to areas of sufficiently high reptile densities to support young snakes., (© 2013 John Wiley & Sons Ltd.)

Bats are well known for species richness and ecological diversity, and thus, they provide a good opportunity to study relationships and interaction between species. To assess interactions, we consider distinct traits that are probably to be triggered by niche shape and evolutionary processes. We present data on the trophic niche differentiation between two sympatric European trawling bat species, Myotis dasycneme and Myotis daubentonii, incorporating a wide spectrum of methodological approaches. We measure morphological traits involved in foraging and prey handling performance including bite force, weightlifting capacity and wing morphology. We then measure resulting prey consumption using both morphological and molecular diet analyses. These species closely resemble each other in morphological traits, however, subtle but significant differences were apparent in bite force and lift capacity, which are related to differences in basic body and head size. Both morphological and molecular diet analyses show strong niche overlap. We detected subtle differences in less frequent prey items, as well as differences in the exploitation of terrestrial and aquatic-based prey groups. Myotis dasycneme feeds more on aquatic prey, like Chironomidae and their pupal stages, or on the aquatic moth Acentria ephemerella. Myotis daubentonii feeds more on terrestrial prey, like Brachycera, or Coleoptera. This suggests that these bats use different microhabitats within the habitat where they co-occur., (© 2013 John Wiley & Sons Ltd.)

Re-introduction of rare species to parts of their historical range is becoming increasingly important as a conservation strategy. Telfair's Skinks (Leiolopisma telfairii), once widespread on Mauritius, were until recently found only on Round Island. There it is vulnerable to stochastic events, including the introduction of alien predators that may either prey upon it or compete for food resources. Consequently, skinks have been introduced to Ile aux Aigrettes, another small Mauritian island that has been cleared of rats. However, the island has been invaded by Asian Musk Shrews (Suncus murinus), a commensal species spread by man well beyond its natural Asian range. Our aim was to use next-generation sequencing to analyse the diets of the shrews and skinks to look for niche competition. DNA was extracted from skink faeces and from the stomach contents of shrews. Application of shrew- and skink-specific primers revealed no mutual predation. The DNA was then amplified using general invertebrate primers with tags to identify individual predators, and then sequenced by 454 pyrosequencing. 119 prey MOTUs (molecular taxonomic units) were isolated, although none could be identified to species. Seeding of cladograms with known sequences allowed higher taxonomic assignments in some cases. Although most MOTUs were not shared by shrews and skinks, Pianka's niche overlap test showed significant prey overlap, suggesting potentially strong competition where food resources are limited. These results suggest that removal of the shrews from the island should remain a priority., (© 2013 John Wiley & Sons Ltd.)

Migration is widespread among vertebrates, yet bat migration has received little attention and only in the recent decades has a better understanding of it been gained. Migration can cause significant changes in behaviour and physiology, due to increasing energy demands and aerodynamic constraints. Dietary shifts, for example, have been shown to occur in birds before onset of migration. For bats, it is not known if a change in diet occurs during migration, although breeding season-related dietary preference has been documented. It is known that a diet rich in fats and the accumulation of fat deposits do increase the flight range of migratory bats. Some bat species can be regarded as long-distance migrants, covering up to 2000 km between summer and winter roosting areas. Pipistrellus nathusii (Vespertilionidae), a European long-distant migrant, travels each year along the Baltic Sea from north-eastern Europe to hibernate in central and southern Europe. This study presents data on the dietary habits of migrating Pipistrellus nathusii compared with those during the breeding season. We analysed faecal samples from bats on fall migration caught at the Ornithological Field Station in Pape, Latvia and from samples collected in North-Latvian summer roosts. We applied both morphological identification and molecular methods, as morphological methods also recognize life stages of prey and can contribute frequency data. The diets of bats on migration and breeding bats were similar, with Diptera and Lepidoptera comprising the major prey categories. However, certain prey groups could be explained by the different hunting habitats exploited during migration vs. summer residence., (© 2013 John Wiley & Sons Ltd.)

Variation in prey resources influences the diet and behaviour of predators. When prey become limiting, predators may travel farther to find preferred food or adjust to existing local resources. When predators are habitat limited, local resource abundance impacts foraging success. We analysed the diet of Myotis lucifugus (little brown bats) from Nova Scotia (eastern Canada) to the Northwest Territories (north-western Canada). This distribution includes extremes of season length and temperature and encompasses colonies on rural monoculture farms, and in urban and unmodified areas. We recognized nearly 600 distinct species of prey, of which ≈30% could be identified using reference sequence libraries. We found a higher than expected use of lepidopterans, which comprised a range of dietary richness from ≈35% early in the summer to ≈55% by late summer. Diptera were the second largest prey group consumed, representing ≈45% of dietary diversity early in the summer. We observed extreme local dietary variability and variation among seasons and years. Based on the species of insects that were consumed, we observed that two locations support prey species with extremely low pollution and acidification tolerances, suggesting that these are areas without environmental contamination. We conclude that there is significant local population variability in little brown bat diet that is likely driven by seasonal and geographical changes in insect diversity, and that this prey may be a good indicator of environment quality., (© 2013 John Wiley & Sons Ltd.)

In this investigation, we use variation in wing morphology, echolocation behaviour, patterns of habitat use and molecular diet analysis to demonstrate that six species of sympatric insectivorous bats in Jamaica show significant differences that could explain resource partitioning among the species. High-intensity echolocating species that used shorter, broadband signals and had shorter, broader wings (Pteronotus macleayii, Pteronotus quadridens, Mormoops blainvillii) foraged most in edge habitats, but differed in timing of peak activity. P. macleayii and M. blainvillii differed in diet, but low sample size precluded diet analysis for P. quadridens. High-intensity echolocating species that used longer, more narrowband signals and had longer, narrower wings (Molossus molossus, Tadarida brasiliensis) foraged most in open areas and differed in diet from the other species. Two disparate species were most active in clutter (dense vegetation). Pteronotus parnellii used high-duty-cycle echolocation apparently specialized for detecting fluttering targets in clutter. Macrotus waterhousii used low-intensity, broadband echolocation calls and presumably uses prey-generated sounds when foraging. These two species also differed in diet. Our data show that differences in morphology and echolocation behaviour coincide with differences in habitat use and diet, resulting in minimal overlap in resource use among species.

Contamination pathways in complex food chains in soil ecosystems can be difficult to elucidate. Molecular analysis of predator gut content can, however, rapidly reveal previously unidentified trophic interactions between invertebrates and thereby uncover pathways of pollutant spread. Here, we measured concentrations of the toxic metals lead, cadmium and mercury in carabid beetle predators and their prey. Invertebrates were sampled at one control and four heavy metal-polluted sites to reveal the impact of diet composition and seasonal variation in prey availability on metal burden in carabids and metal transfer pathways through forest ecosystems. This is the first report, to our knowledge, of carabid diet composition based on PCR analysis of gut contents at the forest community level, rather than in cultivated fields. Extensive screening using group- and species-specific primers revealed that carabids ate primarily earthworms and slugs, as well as smaller numbers of woodlice and springtails. Metal concentrations in carabids correlated with seasonal changes in diet. Mercury accumulated in beetle predators more than in their slug prey. As earthworms, slugs and carabid beetles are the major prey of many birds and mammals, prey-predator transfer and associated toxicity are major risks at mercury-contaminated sites. Carabids may be useful bioindicators for assessing the impact of pollutants on soil ecosystems, as long as species and seasonal factors are taken into account., (© 2013 John Wiley & Sons Ltd.)

Knowledge of the dietary choices and trophic niches of organisms is the key to understanding their roles in ecosystems. In seabird diet studies, prey identification is a difficult challenge, often yielding results with technique-specific biases. Additionally, sampling efforts are often not extensive enough to reveal intrapopulational variation. Immature animals, which may constitute up to 50% of a population, may occupy a significantly different trophic niche to more experienced birds, but this remains largely unexplored. We investigated the diet of Cory's shearwater (Calonectris diomedea) from Selvagem Grande, an island located off the northwest African coast, collecting a total of 698 regurgitate samples over three consecutive breeding seasons. The diet was assessed using two complementary approaches for prey identification: conventional morphological analysis (using fish vertebrae, otoliths and cephalopod beaks) and DNA barcoding of the 16S rRNA mitochondrial gene, in cases where a positive identification could not be retrieved. Species assignments employed BLAST and distance-based methods, as well as direct optimization of the tree length based on unaligned sequences in POY. This method resulted in robust tree estimates and species assignments, showing its potential for DNA barcoding of stomach contents using hypervariable markers such as the 16S. The molecular approach increased taxonomic resolution and revealed an additional 17 taxa. Diet differed significantly according to breeding status, sex, breeding phase (prelaying and chick rearing) and year. Such direct evidence of trophic segregation within the same population has rarely been shown in seabirds and highlights the importance of including such variables in ecosystem-based management approaches., (© 2014 John Wiley & Sons Ltd.)

The slugs of Britain and Ireland form a well-studied fauna of economic importance. They include many widespread European species that are introduced elsewhere (at least half of the 36 currently recorded British species are established in North America, for example). To test the contention that the British and Irish fauna consists of 36 species, and to verify the identity of each, a species delimitation study was conducted based on a geographically wide survey. Comparisons between mitochondrial DNA (COI, 16S), nuclear DNA (ITS-1) and morphology were investigated with reference to interspecific hybridisation. Species delimitation of the fauna produced a primary species hypothesis of 47 putative species. This was refined to a secondary species hypothesis of 44 species by integration with morphological and other data. Thirty six of these correspond to the known fauna (two species in Arion subgenus Carinarion were scarcely distinct and Arion (Mesarion) subfuscus consisted of two near-cryptic species). However, by the same criteria a further eight previously undetected species (22% of the fauna) are established in Britain and/or Ireland. Although overlooked, none are strictly morphologically cryptic, and some appear previously undescribed. Most of the additional species are probably accidentally introduced, and several are already widespread in Britain and Ireland (and thus perhaps elsewhere). At least three may be plant pests. Some evidence was found for interspecific hybridisation among the large Arion species (although not involving A. flagellus) and more unexpectedly in species pairs in Deroceras (Agriolimacidae) and Limacus (Limacidae). In the latter groups, introgression appears to have occurred in one direction only, with recently-invading lineages becoming common at the expense of long-established or native ones. The results show how even a well-studied, macroscopic fauna can be vulnerable to cryptic and undetected invasions and changes.

Predicting whether a predator is capable of affecting the dynamics of a prey species in the field implies the analysis of the complete diet of the predator, not simply rates of predation on a target taxon. Here, we employed the Ion Torrent next-generation sequencing technology to investigate the diet of a generalist arthropod predator. A complete dietary analysis requires the use of general primers, but these will also amplify the predator unless suppressed using a blocking probe. However, blocking probes can potentially block other species, particularly if they are phylogenetically close. Here, we aimed to demonstrate that enough prey sequence could be obtained without blocking probes. In communities with many predators, this approach obviates the need to design and test numerous blocking primers, thus making analysis of complex community food webs a viable proposition. We applied this approach to the analysis of predation by the linyphiid spider Oedothorax fuscus in an arable field. We obtained over two million raw reads. After discarding the low-quality and predator reads, the libraries still contained over 61 000 prey reads (3% of the raw reads; 6% of reads passing quality control). The libraries were rich in Collembola, Lepidoptera, Diptera and Nematoda. They also contained sequences derived from several spider species and from horticultural pests (aphids). Oedothorax fuscus is common in UK cereal fields, and the results showed that it is exploiting a wide range of prey. Next-generation sequencing using general primers but without blocking probes provided ample sequences for analysis of the prey range of this spider and proved to be a simple and inexpensive approach., (© 2013 John Wiley & Sons Ltd.)

The dynamics of predation on parasites within prey has received relatively little attention despite the profound effects this is likely to have on both prey and parasite numbers and hence on biological control programmes where parasites are employed. The nematode Phasmarhabditis hermaphrodita is a commercially available biological agent against slugs. Predation on these slugs may, at the same time, result in intraguild predation on slug-parasitic nematodes. This study describes, for the first time, predation by carabid beetles on slugs and their nematode parasites on both spatial and temporal scales, using PCR-based methods. The highest nematode infection levels were found in the slugs Deroceras reticulatum and Arion silvaticus. Numbers of infected slugs decreased over time and no infected slugs were found four months after nematode application. The density of the most abundant slug, the invasive Arion vulgaris, was positively related to the activity-density of the carabid beetle, Carabus nemoralis. Predation on slugs was density and size related, with highest predation levels also on A. vulgaris. Predation on A. vulgaris decreased significantly in summer when these slugs were larger than one gram. Predation by C. nemoralis on slugs was opportunistic, without any preferences for specific species. Intraguild predation on the nematodes was low, suggesting that carabid beetles such as C. nemoralis probably do not have a significant impact on the success of biological control using P. hermaphrodita.

Aphids are major pests in apple orchards, debilitating the crop and spreading disease. We investigated whether early-season predation by canopy spiders may be effectively controlling aphid numbers in three organic orchards. For this purpose, we monitored the aphid population dynamics from the winter eggs to colony stages and compared this to spider abundances and rates of predation on aphids detected by diagnostic polymerase chain reaction. For the latter, we applied existing general aphid primers. We found that spiders ate colony fundatrices and that aphid numbers were negatively related to spider abundance. Spiders were the main active predators within the orchards when the first colony fundatrices were present, indicating their importance in the early control of aphid populations.

The whiteflies Bemisia tabaci Gennadius and Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) are two of the main pests in tomato crops. Their biological control in Mediterranean IPM systems is based on the predators Macrolophus pygmaeus (Rambur) and Nesidiocoris tenuis Reuter (Hemiptera: Miridae), as well as on the parasitoids Eretmocerus mundus (Mercet) and Encarsia pergandiella Howard (Hymenoptera: Aphelinidae). These natural enemies may interact with each other and their joint use could interfere with the biological control of those whitefly pests. Analysis of predator-prey interactions under field conditions is therefore essential in order to optimize whitefly control. Species-specific polymerase chain reaction (PCR)-primers were designed to detect DNA fragments of these whiteflies and parasitoids within both predator species in tomato greenhouses. We demonstrated that both predators feed on both whitefly species, as well as on both parasitoids under greenhouse conditions. Prey molecular detection was possible where prey abundance was very low or even where predation was not observed under a microscope. Whitefly DNA detection was positively correlated with adult whitefly abundance in the crop. However, a significant relationship was not observed between parasitoid DNA detection and the abundance of parasitoid pupae, even though the predation rate on parasitoids was high. This unidirectional intraguild predation (predators on parasitoids) could potentially reduce their combined impact on their joint prey/host. Prey molecular detection provided improved detection of prey consumption in greenhouse crops, as well as the possibility to identify which prey species were consumed by each predator species present in the greenhouse, offering a blueprint with wider applicability to other food webs.

The molecular detection of predation is a fast growing field, allowing highly specific and sensitive detection of prey DNA within the gut contents or faeces of a predator. Like all molecular methods, this technique is prone to potential sources of error that can result in both false positive and false negative results. Here, we test the hypothesis that the use of suction samplers to collect predators from the field for later molecular analysis of predation will lead to high numbers of false positive results. We show that, contrary to previous published work, the use of suction samplers resulted in previously starved predators testing positive for aphid and collembolan DNA, either as a results of ectopic contamination or active predation in the collecting cup/bag. The contradictory evidence for false positive results, across different sampling protocols, sampling devices and different predator-prey systems, highlights the need for experimentation prior to mass field collections of predators to find techniques that minimise the risk of false positives.

Generalist predators and parasitoids are considered to be important regulators of aphids. The former not only feed on these pests, but might also consume parasitoids at all stages of development. This direct or coincidental interference affects the natural control of aphids, the scale of which is largely unknown, and it has rarely been examined under natural conditions. Here, molecular diagnostics were used to track trophic interactions in an aphid-parasitoid-generalist predator community during the build-up of a cereal aphid population. We found that generalist predators, principally carabid and staphylinid beetles as well as linyphiid spiders, had strong trophic links to both parasitoids and aphids. Remarkably, more than 50% of the parasitoid DNA detected in predators stems from direct predation on adult parasitoids. The data also suggest that coincidental intraguild predation is common too. Generalist predators, hence, disrupt parasitoid aphid control, although the levels at which the predators feed on pests and parasitoids seem to vary significantly between predator taxa. Our results suggest that taxon-specific trophic interactions between natural enemies need to be considered to obtain a more complete understanding of the route to effective conservation biological control.

The analysis of food webs and their dynamics facilitates understanding of the mechanistic processes behind community ecology and ecosystem functions. Having accurate techniques for determining dietary ranges and components is critical for this endeavour. While visual analyses and early molecular approaches are highly labour intensive and often lack resolution, recent DNA-based approaches potentially provide more accurate methods for dietary studies. A suite of approaches have been used based on the identification of consumed species by characterization of DNA present in gut or faecal samples. In one approach, a standardized DNA region (DNA barcode) is PCR amplified, amplicons are sequenced and then compared to a reference database for identification. Initially, this involved sequencing clones from PCR products, and studies were limited in scale because of the costs and effort required. The recent development of next generation sequencing (NGS) has made this approach much more powerful, by allowing the direct characterization of dozens of samples with several thousand sequences per PCR product, and has the potential to reveal many consumed species simultaneously (DNA metabarcoding). Continual improvement of NGS technologies, on-going decreases in costs and current massive expansion of reference databases make this approach promising. Here we review the power and pitfalls of NGS diet methods. We present the critical factors to take into account when choosing or designing a suitable barcode. Then, we consider both technical and analytical aspects of NGS diet studies. Finally, we discuss the validation of data accuracy including the viability of producing quantitative data., (© 2011 Blackwell Publishing Ltd.)

Little quantitative ecological information exists on the diets of most invertebrate feeding reptiles, particularly nocturnal or elusive species that are difficult to observe. In the UK and elsewhere, reptiles are legally required to be relocated before land development can proceed, but without knowledge of their dietary requirements, the suitability of receptor sites cannot be known. Here, we tested the ability of non-invasive DNA-based molecular diagnostics (454 pyrosequencing) to analyse reptile diets, with the specific aims of determining which earthworm species are exploited by slow worms (the legless lizard Anguis fragilis) and whether they feed on the deeper-living earthworm species that only come to the surface at night. Slow worm faecal samples from four different habitats were analysed using earthworm-specific PCR primers. We found that 86% of slow worms (N=80) had eaten earthworms. In lowland heath and marshy/acid grassland, Lumbricus rubellus, a surface-dwelling epigeic species, dominated slow worm diet. In two other habitats, riverside pasture and calciferous coarse grassland, diet was dominated by deeper-living anecic and endogeic species. We conclude that all species of earthworm are exploited by these reptiles and lack of specialization allows slow worms to thrive in a wide variety of habitats. Pyrosequencing of prey DNA in faeces showed promise as a practical, rapid and relatively inexpensive means of obtaining detailed and valuable ecological information on the diets of reptiles., (© 2011 Blackwell Publishing Ltd.)

Relatively few studies have examined the parasite fauna of British reptiles, partly due to the cryptic nature and low population density of these hosts. Here we examined 12 populations of the slow worm Anguis fragilis which, unlike other UK lizards, occurs at locally high population densities. Morphological examination of non-invasively collected faecal samples revealed the presence of Neoxysomatium brevicaudatum and a second unidentified nematode species. Although previously unrecorded from slow worms in the UK, N. brevicaudatum was present in 38% of animals (mean intensity 70.9, range 1-686). Morphological identification was confirmed by sequencing the 18S ribosomal gene. The use of the species-specific, cytochrome oxidase I mitochondrial gene primers proved an efficient alternative to conventional, microscope screening for parasites, although the original identification of N. brevicaudatum was dependent upon morphological characters. Sequencing also identified the second, smaller nematode as belonging to the Rhabdiasidae family: this species was even more common at a prevalence of 83% (mean intensity 102.8, range 1-2000). While increasing our knowledge of the UK macroparasite fauna, this work demonstrates the benefits of a combined morphological-molecular approach.

Molecular sexing revealed an unexpectedly strong female bias in the sex ratio of pre-breeding European Storm Petrels (Hydrobates pelagicus), attracted to playback of conspecific calls during their northwards migration past SW Europe. This bias was consistent across seven years, ranging from 80.8% to 89.7% female (mean annual sex ratio ± SD = 85.5% female ±4.1%). The sex ratio did not differ significantly from unity (i.e., 50% female) among (i) Storm Petrel chicks at a breeding colony in NW France, (ii) adults found dead on beaches in Southern Portugal, (iii) breeding birds attending nest burrows in the UK, captured by hand, and (iv) adults captured near a breeding colony in the UK using copies of the same sound recordings as used in Southern Europe, indicating that females are not inherently more strongly attracted to playback calls than males. A morphological discriminant function analysis failed to provide a good separation of the sexes, showing the importance of molecular sexing for this species. We found no sex difference in the seasonal or nocturnal timing of migration past Southern Europe, but there was a significant tendency for birds to be caught in sex-specific aggregations. The preponderance of females captured in Southern Europe suggests that the sexes may differ in migration route or in their colony-prospecting behaviour during migration, at sites far away from their natal colonies. Such differences in migration behaviour between males and females are poorly understood but have implications for the vulnerability of seabirds to pollution and environmental change at sea during the non-breeding season.

The invasive Iberian slug, Arion lusitanicus, is spreading through Europe and poses a major threat to horticulture and agriculture. Natural enemies, capable of killing A. lusitanicus, may be important to our understanding of its population dynamics in recently invaded regions. We used polymerase chain reaction (PCR) to study predation on A. lusitanicus by carabid beetles in the field. A first multiplex PCR was developed, incorporating species-specific primers, and optimised in order to amplify parts of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of large Arion slugs, including A. lusitanicus from the gut contents of the predators. A second multiplex PCR, targeting 12S rRNA mtDNA, detected predation on smaller Arion species and the field slug Deroceras reticulatum. Feeding trials were conducted to measure the effects of digestion time on amplicon detectability. The median detection times (the time at which 50% of samples tested positive) for A. lusitanicus and D. reticulatum DNA in the foreguts of Carabus nemoralis were 22 h and 20 h, respectively. Beetle activity-densities were monitored using pitfall traps, and slug densities were estimated using quadrats. Predation rates on slugs in the field by C. nemoralis in spring ranged from 16-39% (beetles positive for slug DNA) and were density dependent, with numbers of beetles testing positive being positively correlated with densities of the respective slug species. Carabus nemoralis was shown to be a potentially important predator of the alien A. lusitanicus in spring and may contribute to conservation biological control.

Primers were developed for leafhoppers of the genus Aphrodes amplifying 84-244 bp fragments of the mitochondrial cytochrome oxidase subunit I gene. DNA was extracted from legs of over 100-year-old archived museum specimens, amplified and sequenced. The fragments contain sufficient variation to unequivocally identify the different species. The majority of the analysed museum specimens, including three specimens of the syntype series for the UK endemic species A. aestuarina (Edwards 1908), were found to have been assigned to the wrong species. This work clearly underlines the need to validate museum specimens using molecular methods where identity is in doubt, based on reliable standards for species discrimination., (© 2011 Blackwell Publishing Ltd.)

Vibrational signalling is a widespread form of animal communication and, in the form of sexual communication, has been generally regarded as inherently short-range and a private communication channel, free from eavesdropping by generalist predators. A combination of fieldwork and laboratory experiments was used to test the hypothesis that predators can intercept and exploit such signals. First, we developed and characterized PCR primers specific for leafhoppers of the genus Aphrodes and specifically for the species Aphrodes makarovi. Spiders were collected from sites where leafhoppers were present and screened with these primers to establish which spider species were significant predators of this species during the mating period of these leafhoppers. Analysis using PCR of the gut contents of tangle-web spiders, Enoplognatha ovata (Theridiidae), showed that they consume leafhoppers in the field at a greater rate when signalling adults were present than when nymphs were dominant, suggesting that the spiders were using these vibrations signals to find their prey. Playback and microcosm experiments then showed that E. ovata can use the vibrational signals of male leafhoppers as a cue during foraging and, as a result, killed significantly more male than female A. makarovi. Our results show, for the first time, that arthropod predators can exploit prey vibrational communication to obtain information about prey availability and use this information to locate and capture prey. This may be a widespread mechanism for prey location, one that is likely to be a major unrecognized driver of evolution in both predators and prey., (© 2011 Blackwell Publishing Ltd.)

Landscape management affects species interactions within a community, leading to alterations in the structure of networks. Modules are link-dense regions of the network where species interact more closely within the module than between modules of the network. Insufficient network resolution has meant that modules have proved difficult to identify, even though they appear important in the propagation of disturbance impacts. We applied a standardized approach across 20 farms to obtain well-resolved food webs to characterize network structure and explore how modularity changes in response to management (organic and conventional). All networks showed significantly higher modularity than random networks. Farm management had no effect on the number of modules per farm or module richness, but there was a significant loss of links between modules on conventional farms, which may affect the long-term stability of these networks. We found a significant association between modules and major habitat groups. If modules form as a result of interactions between species that utilize similar habitats, then ecosystem services to the crop components of the landscape, such as pest control by parasitoids originating in the non-crop vegetation, are less likely to occur on these farms.

Species- and group-specific PCR primers were developed to study predation on pest and nonpest invertebrate species by generalist carabid predators in agroecosystems. To ensure the amplification of degraded DNA in predator gut samples, amplicons were designed to be less than 300 bp. Specificity of primers was assessed by cross-amplification against a panel of target and nontarget invertebrate species. The new primers were combined with previously published primers for slugs and collembolla in multiplex reactions to simultaneously screen each predator for the presence of multiple prey. All prey species were detected in a screen of the gut contents of field-caught predators., (© 2010 Blackwell Publishing Ltd.)

Arion lusitanicus has become a major pest species in western Norway in the last few years. This species originates from southern Europe but has been spread by humans over large parts of central and northern Europe during recent decades. Slugs have traditionally been controlled by the use of molluscicides; but, as these may have serious ecological side effects, biological control of slugs is highly desirable. Potential biological control agents include nematodes, gastropods and arthropods. In laboratory experiments, we tested whether five common predator beetles would feed on eggs and juveniles of A. lusitanicus. The species Carabus nemoralis, Nebria brevicollis, Pterostichus melanarius and Pterostichus niger (Carabidae) as well as Staphylinus erythropterus (Staphylinidae) were tested, of which only P. melanarius has been tested on A. lusitanicus previously. Nebria brevicollis did not feed on slug eggs or newly hatched slugs, but the remaining four species all killed and ate a large proportion of the eggs and hatchlings offered. Both P. melanarius and P. niger also destroyed A. lusitanicus eggs and hatchlings under conditions emulating those in the field. Prey size choice experiments were conducted by feeding C. nemoralis, P. niger and S. erythropterus on different sizes of A. lusitanicus. Carabus nemoralis was also given a choice between two slug species, A. lusitanicus and Deroceras reticulatum. A significant preference for slugs smaller than one gram was evident for C. nemoralis, while the other beetles struggled much more to overcome the mucus of juvenile slugs. No significant preference was found between A. lusitanicus and D. reticulatum as prey for C. nemoralis. We also discuss the feasibility of biological control of A. lusitanicus using beetle predators.

The carabid beetle Pterostichus melanarius is a major natural enemy of pests, such as aphids and slugs in agricultural systems. Earthworms are a dominant non-pest component of the diet of P. melanarius which help sustain the beetles during periods when the pest population is low or absent. In this study we wanted to test whether this predator exercises prey choice among different earthworm species or ecological groups. High levels of genetic diversity within morphological species of earthworm necessitated the development of primers that were specific not just to species but lineages and sub-lineages within species as well. Gut samples from beetles were analysed using multiplex-PCR and fluorescent-labelled primers. Calibratory feeding trials were undertaken to calculate median detection times for prey DNA following ingestion. Extensive testing demonstrated that the primers were species-specific, that detection periods were negatively related to amplicon size and that meal size had a highly significant effect on detection periods. Monte Carlo simulations showed that, in general, worms were being predated in proportion to their densities in the field with little evidence of prey choice, other than probable avoidance of the larger, deep-living species. There was no evidence that epigeic species were being taken preferentially in comparison with endogeic species. There was also no evidence that defensive secretions by Allolobophora chlorotica reduced predation pressure on this species by P. melanarius. We concluded that any management system that increases earthworm densities generally, regardless of component species, is likely to be optimal for increasing numbers of this beneficial beetle predator.

While many studies have demonstrated that organic farms support greater levels of biodiversity, it is not known whether this translates into better provision of ecosystem services. Here we use a food-web approach to analyse the community structure and function at the whole-farm scale. Quantitative food webs from 10 replicate pairs of organic and conventional farms showed that organic farms have significantly more species at three trophic levels (plant, herbivore and parasitoid) and significantly different network structure. Herbivores on organic farms were attacked by more parasitoid species on organic farms than on conventional farms. However, differences in network structure did not translate into differences in robustness to simulated species loss and we found no difference in percentage parasitism (natural pest control) across a variety of host species. Furthermore, a manipulative field experiment demonstrated that the higher species richness of parasitoids on the organic farms did not increase mortality of a novel herbivore used to bioassay ecosystem service. The explanation for these differences is likely to include inherent differences in management strategies and landscape structure between the two farming systems.

Aphid predators are a systematically disparate group of arthropods united on the basis that they consume aphids as part of their diet. In Europe, this group includes Araneae, Opiliones, Heteroptera, chrysopids, Forficulina, syrphid larvae, carabids, staphylinids, cantharids and coccinellids. This functional group has no phylogenetic meaning but was created by ecologists as a way of understanding predation, particularly for conservation biological control. We investigated whether trait-based approaches could bring some cohesion and structure to this predator group. A taxonomic hierarchy-based null model was created from taxonomic distances in which a simple multiplicative relationship described the Linnaean hierarchies (species, genera, etc.) of fifty common aphid predators. Using the same fifty species, a functional groups model was developed using ten behavioural traits (e.g. polyphagy, dispersal, activity, etc.) to describe the way in which aphids were predated in the field. The interrelationships between species were then expressed as dissimilarities within each model and separately analysed using PROXSCAL, a multidimensional scaling (MDS) program. When ordinated using PROXSCAL and then statistically compared using Procrustes analysis, we found that only 17% of information was shared between the two configurations. Polyphagy across kingdoms (i.e. predatory behaviour across animal, plant and fungi kingdoms) and the ability to withstand starvation over days, weeks and months were particularly divisive within the functional groups model. Confirmatory MDS indicated poor prediction of aphid predation rates by the configurations derived from either model. The counterintuitive conclusion was that the inclusion of functional traits, pertinent to the way in which predators fed on aphids, did not lead to a large improvement in the prediction of predation rate when compared to the standard taxonomic approach.

Earthworms play a major role in many aspects of soil fertility, food web ecology and ecosystem functioning, and hence are frequently the subjects of, for example, ecological and toxicological research. Our aim was to examine the genetic structure of common earthworm species, to identify cryptic lineages or species that may be distinct ecotypes or biotypes (and hence confound current research based upon morphotypes) and to try to explain the massive cryptic diversity that eventually emerged. We demonstrated that species such as Allolobophora chlorotica, Aporrectodea longa, Aporrectodea rosea and Lumbricus rubellus all comprise highly divergent lineages with species-level divergence at the mitochondrial cytochrome oxidase I (COI) gene. In Allo. chlorotica alone, we found 55 haplotypes for COI, with 35 of these being found in pink and 20 in green morph worms. There were no cases of the two colour morphs sharing COI haplotypes. Phylogenetic analyses of mitochondrial COI and 16S genes showed the presence of five highly divergent lineages, suggesting the presence of multiple cryptic species within Allo. chlorotica. There was no clear geographical pattern to lineage distribution and many populations were polymorphic for both mitochondrial DNA lineage and colour morph. Amplified fragment length polymorphism results, based on two primer combinations, were broadly congruent with mitochondrial DNA results with one significant exception. Despite showing over 14% divergence at COI, amplified fragment length polymorphism markers showed that the two green morph lineages may be interbreeding and therefore represent a single taxon. The cryptic diversity revealed by these results has profound consequences for all areas of earthworm research.

Insect parasitoids play a major role in terrestrial food webs as they are highly diverse, exploit a wide range of niches and are capable of affecting host population dynamics. Formidable difficulties are encountered when attempting to quantify host-parasitoid and parasitoid-parasitoid trophic links in diverse parasitoid communities. Here we present a DNA-based approach to effectively track trophic interactions within an aphid-parasitoid food web, targeting, for the first time, the whole community of parasitoids and hyperparasitods associated with a single host. Using highly specific and sensitive multiplex and singleplex polymerase chain reaction, endoparasitism in the grain aphid Sitobion avenae (F) by 11 parasitoid species was quantified. Out of 1061 aphids collected during 12 weeks in a wheat field, 18.9% were found to be parasitized. Parasitoids responded to the supply of aphids, with the proportion of aphids parasitized increasing monotonically with date, until the aphid population crashed. In addition to eight species of primary parasitoids, DNA from two hyperparasitoid species was detected within 4.1% of the screened aphids, with significant hyperparasitoid pressure on some parasitoid species. In 68.2% of the hyperparasitized aphids, identification of the primary parasitoid host was also possible, allowing us to track species-specific parasitoid-hyperparasitoid links. Nine combinations of primary parasitoids within a single host were found, but only 1.6% of all screened aphids were multiparasitized. The potential of this approach to parasitoid food web research is discussed.

The effects of predators on prey populations can be modified by a number of abiotic factors. Here, we investigated the combined and separate effects of rain and ground-dwelling predators on aphid populations in a microcosm experiment lasting for 21 days, using PCR to analyse the gut content of the predators. Rain significantly dislodged aphids from shoots and ears by 57% and 25%, respectively. The gut content analysis showed that more predators consumed aphids in the rain treatment than without rain, indicating higher availability of aphids to ground-dwelling predators after rain. However, no synergistic effects of rain and ground-dwelling predators on aphid population development could be demonstrated. Rain alone significantly decreased aphid populations by 27%, suggesting that this is a significant mortality factor. Predators alone had no significant effect on aphid numbers, but the gut content analyses showed aphid consumption also in the no-rain treatments, indicating that aphids were available to the predators on the soil surface even without rain. Our results suggest that weather conditions such as rain can modify predator-prey interactions in the field. Employing PCR-based predator gut content analyses proved to be useful as trophic links could be directly verified.

PCR-based techniques to investigate predator-prey trophic interactions are starting to be used more widely, but factors affecting DNA decay in predator guts are still poorly understood. Here, we investigated the effects of time since feeding, temperature and amplicon size on the detectability of prey DNA in the gut content of two closely related predator species. Cereal aphids, Sitobion avenae, were fed to the carabid beetles Pterostichus melanarius and Nebria brevicollis. Beetles were allowed to digest their meal at 12 degrees C, 16 degrees C and 20 degrees C, and batches of beetles were subsequently frozen at time periods from 0-72 h after feeding. Aphid DNA was detected within beetles' gut contents using primers amplifying fragments of 85, 231, 317 and 383 bp. Prey DNA detection rates were significantly higher in N. brevicollis than in P. melanarius, indicating fundamental dissimilarities in prey digestion capacities. High temperatures (20 degrees C) and large amplicons (383 bp) significantly decreased detection rates. The shortest amplicon gave the highest prey DNA detection success, whereas no differences were observed between the 231 bp and the 317 bp fragment. Our results indicate that factors such as ambient temperature, predator taxon and amplicon size should all be considered when interpreting data derived from PCR-based prey detection. Correction for such factors should make calculation of predation rates in the field more accurate and could help us to estimate when predation events occur in the field.

Although the effects of nutrient enhancement on aquatic systems are well documented, the consequences of nutritional supplements on soil food webs are poorly understood, and results of past research examining bottom-up effects are often conflicting. In addition, many studies have failed to separate the effects of nutrient enrichment and the physical effects of adding organic matter. In this field study, we hypothesised that the addition of nitrogen to soil would result in a trophic cascade, through detritivores (Collembola) to predators (spiders), increasing invertebrate numbers and diversity. Nitrogen and lime were added to plots in an upland grassland in a randomised block design. Populations of Collembola and spiders were sampled by means of pitfall traps and identified to species. Seventeen species of Collembola were identified from the nitrogen plus lime (N+L) and control plots. Species assemblage, diversity, richness, evenness and total number were not affected by nutrient additions. However, there was an increase in the number of Isotomidae juveniles and Parisotoma anglicana trapped in the N+L plots. Of the 44 spider species identified, over 80% were Linyphiidae. An effect on species assemblage from the addition of N+L to the plots was observed on two of the four sampling dates (July 2002 and June 2003). The linyphiid, Oedothorax retusus, was the only species significantly affected by the treatments and was more likely to be trapped in the control plots.The increased number of juvenile Collembola, and change in community composition of spiders, were consequences of the bottom-up effect caused by nutrient inputs. However, despite efforts to eliminate the indirect effects of nutrient inputs, a reduction in soil moisture in the N+L plots cannot be eliminated as a cause of the invertebrate population changes observed. Even so, this experiment was not confounded by the physical effects of habitat structure reported in most previous studies. It provides evidence of moderate bottom-up influences of epigeic soil invertebrate food webs and distinguishes between nutrient addition and plant physical structure effects. It also emphasises the importance of understanding the effects of soil management practices on soil biodiversity, which is under increasing pressure from land development and food production.

Predation on parasitized hosts can significantly affect natural enemy communities, and such intraguild predation may indirectly affect control of herbivore populations. However, the methodological challenges for studying these often complex trophic interactions are formidable. Here, we evaluate a DNA-based approach to track parasitism and predation on parasitized hosts in model herbivore-parasitoid-predator systems. Using singleplex polymerase chain reaction (SP-PCR) to target mtDNA of the parasitoid only, and multiplex PCR (MP-PCR) to additionally target host DNA as an internal amplification control, we found that detection of DNA from the parasitoid, Lysiphlebus testaceipes, in its aphid host, Aphis fabae, was possible as early as 5 min. post parasitism. Up to 24 h post parasitism SP-PCR proved to be more sensitive than MP-PCR in amplifying parasitoid DNA. In the carabid beetles Demetrias atricapillus and Erigone sp. spiders, fed with aphids containing five-day-old parasitoids, parasitoid and aphid DNA were equally detectable in both predator groups. However, when hosts containing two-day-old parasitoids were fed to the predators, detection of parasitoid prey was possible only at 0 h (immediately after consumption) and up to 8 h post consumption in carabids and spiders, respectively. Over longer periods of time, post-feeding prey detection success was significantly higher in spiders than in carabid beetles. MP-PCR, in which parasitoid and aphid DNA were simultaneously amplified, proved to be less sensitive at amplifying prey DNA than SP-PCR. In conclusion, our study demonstrates that PCR-based parasitoid and prey detection offers an exciting approach to further our understanding of host-parasitoid-predator interactions.

Spatially explicit predator-prey interactions can alter the predatory potential of natural enemies augmented through conservation biological control. To test hypotheses regarding such interactions and predatory efficiency, we used a combination of molecular techniques and mark-release-recapture to study the foraging behaviour of a generalist carabid predator, Poecilus cupreus, in response to spatial patterns of its cereal aphid prey (Metapolophium dirhodum and Sitobion avenae). Beetle and aphid numbers were measured across two grids of sampling locations, within which aphid spatial pattern had been manipulated to generate patchy and more homogenous distributions. Aphid consumption was measured by enzyme-linked immunosorbent assays (ELISA) of beetle gut contents, using an aphid-specific monoclonal antibody. Movement and distribution patterns suggest that P. cupreus does not aggregate at, nor instigate prey-taxis within, aphid patches. However, more than two-thirds of the 2169 P. cupreus tested by ELISA had consumed aphids and the proportion of beetles containing aphid proteins was positively related to aphid density. Against expectation, the proportion of predators feeding on aphids was greatest where prey were homogenously distributed, and this was attributed to the loss of partial refuges for prey in aphid patches. The functional value of this type of uniform foraging strategy is ideally suited to early colonization of the crop habitat, when aphid numbers are low, before populations build up and form strong spatial patterns.

Molecular analysis of predation, through polymerase chain reaction amplification of prey remains within the faeces or digestive systems of predators, is a rapidly growing field, impeded by a lack of readily accessible advice on best practice. Here, we review the techniques used to date and provide guidelines accessible to those new to this field or from a different molecular biology background. Optimization begins with field collection, sample preservation, predator dissection and DNA extraction techniques, all designed to ensure good quality, uncontaminated DNA from semidigested samples. The advantages of nuclear vs. mitochondrial DNA as primer targets are reviewed, along with choice of genes and advice on primer design to maximize specificity and detection periods following ingestion of the prey by the predators. Primer and assay optimization are discussed, including cross-amplification tests and calibratory feeding experiments. Once primers have been made, the screening of field samples must guard against (through appropriate controls) cross contamination. Multiplex polymerase chain reactions provide a means of screening for many different species simultaneously. We discuss visualization of amplicons on gels, with and without incorporation of fluorescent primers. In more specialized areas, we examine the utility of temperature and denaturing gradient gel electrophoresis to examine responses of predators to prey diversity, and review the potential of quantitative polymerase chain reaction systems to quantify predation. Alternative routes by which prey DNA might get into the guts of a predator (scavenging, secondary predation) are highlighted. We look ahead to new technologies, including microarrays and pyrosequencing, which might one day be applied to this field.

Environment-friendly farming techniques seek to increase invertebrate biodiversity in part with the intention of encouraging greater numbers of predators that will help to control crop pests. However, in theory, this effect may be negated if the availability of a greater abundance and diversity of alternative prey diverts predators away from feeding on pests. The hypothesis that access to alternative prey can lead to reduced pest suppression under semi-field conditions was tested. Alternative prey type and diversity were manipulated in 70 mesocosms over 7+ weeks in the presence of the carabid Pterostichus melanarius (Illiger), a known predator of slugs, and reproducing populations of the slug Deroceras reticulatum (Müller). Significantly fewer slugs survived where no alternative prey were provided. Maximum slug numbers and biomass were found in treatments containing either carabids plus a high diversity of alternative prey (many species of earthworm and three of Diptera larvae) or a single additional prey (blowfly larvae, Calliphora vomitoria Linnaeus). In these treatments slug numbers and biomass were as high as in plots lacking predators. The effects of alternative prey were taxon-specific. Alternative prey strongly affected carabid fitness in terms of biomass and egg load. The fittest predators (those with access to high alternative prey diversity or C. vomitoria larvae) reduced slug numbers the least. The mean individual slug weights were greater in treatments with alternative prey than where no alternative prey were provided to the carabids. These results suggest that pests may survive and reproduce more rapidly in patches where predators have access to alternative prey.

The relative importance of the factors driving change in the population dynamics of nematodes in the soil is almost completely unknown. Top-down control by micro-arthropod predators may have a significant impact on nematode population dynamics. We report experiments showing that mites and Collembola were capable of reducing nematode numbers in the laboratory and were feeding on a targeted nematode species in the field. A PCR-based approach was developed for the detection of predation on three species of slug- and insect-pathogenic nematodes: Phasmarhabditis hermaphrodita, Heterorhabditis megidis and Steinernema feltiae. The collembolan Folsomia candida and the mesostigmatid mite Stratiolaelaps miles were employed as model predators to calibrate post-ingestion prey DNA detection times. Fragments of cytochrome oxidase I (COI) mtDNA were sequenced and species-specific primers were designed, amplifying 154-, 154- and 203-bp fragments for each of the nematode species. Detection times for nematode DNA within the guts of Collembola were longer than in mites, with half-lives (50% of samples testing positive) of 08.75 h and 05.03 h, respectively. F. candida significantly reduced numbers of the nematode H. megidis, with rates of predation of approximately 0.4 nematode infective juveniles per collembolan per hour over 10 h. Four taxa of field-caught micro-arthropod that had been exposed to the nematode P. hermaphrodita for a period of 12 h were analysed and significant numbers of three taxa tested positive. This is the first application of PCR techniques for the study of nematophagy and the first time these techniques have been used to measure predation on nematodes in the field.

The utility of temperature gradient gel electrophoresis (TGGE) as a means of analysing the gut contents of predators was evaluated. Generalist predators consume multiple prey species and a species-specific primer approach may not always be a practical means of analysing predator responses to prey diversity in complex and biodiverse ecosystems. General invertebrate primers were used to amplify the gut contents of predators, generating banding patterns that identified component prey remains. There was no evidence of dominance of the polymerase chain reaction (PCR) by predator DNA. When applied to field samples of the carabid predator Pterostichus melanarius (Illiger) nine banding patterns were detected, including one for aphids. To further distinguish between species, group-specific primers were designed to separate species of earthworm and aphid. TGGE of the earthworm PCR products generated banding patterns that varied with haplotype in some species. Aphid and earthworm DNA could be detected in the guts of carabids for up to 24 h using TGGE. In P. melanarius, with low numbers of prey per insect gut (mean<3), interpretation of banding patterns proved to be tractable. Potential problems of interpretation of TGGE gels caused by multiple prey bands, cryptic bands, haplotype variation, taxonomic uncertainties (especially with regard to earthworms), secondary predation, scavenging and presence of parasites and parasitoids in the prey or the predators, are discussed. The results suggest that PCR, using combinations of general invertebrate and group-specific primers followed by TGGE, provides a potentially useful approach to the analysis of multiple uncharacterized prey in predators.

Predation by generalist predators is difficult to study in the field because of the complex effects of positive and negative interactions within and between predator species and guilds. Predation can be monitored by molecular means, through identification of prey DNA within predators. However, polymerase chain reaction (PCR) amplification of prey DNA from predators cannot discriminate between primary and secondary predation (hyperpredation), in which one predator feeds on another that has recently eaten the target prey. Here we quantify, for the first time, the potential error caused by detection of prey DNA following secondary predation, using an aphid-spider-carabid model. First, the aphid Sitobion avenae was fed to the spider Tenuiphantes tenuis and the carabid Pterostichus melanarius, and the postconsumption detection periods, for prey DNA within predators, were calculated. Aphids were then fed to spiders and the spiders to carabids. Aphid DNA was detected in the predators using primers that amplified 245- and 110-bp fragments of the mitochondrial cytochrome oxidase I gene. Fragment size and predator sex had no significant effect on detection periods. Secondary predation could be detected for up to 8 h, when carabids fed on spiders immediately after the latter had consumed aphids. Beetles tested positive up to 4 h after eating spiders that had digested their aphid prey for 4 h. Clearly, the extreme sensitivity of PCR makes detection of secondary predation more likely, and the only reliable answer in future may be to use PCR to identify, in parallel, instances of intraguild predation.

Gut-content analyses using molecular techniques are an effective approach to quantifying predator-prey interactions. Predation is often assumed but scavenging is an equally likely route by which animal DNA enters the gut of a predator/scavenger. We used PCR (polymerase chain reaction) to detect scavenged material in predator gut homogenates. The rates at which DNA in decaying slugs (Mollusca: Pulmonata) and aphids (Homoptera: Aphididae) became undetectable were estimated. The detectability of DNA from both carrion types in the guts of the generalist predator Pterostichus melanarius (Coleoptera: Carabidae) was then determined. The effects of carrion age and weight, as well as beetle sex, on detection periods, were quantified. Laboratory trials measured prey preference of beetles between live and decaying prey. Further experiments measured, for the first time, feeding by P. melanarius on dead slugs and aphids directly in the field. In both field and laboratory, P. melanarius preferentially fed on dead prey if available, but preference changed as the prey became increasingly decayed. Disappearance rates for slug carrion in wheat fields and grasslands were estimated and P. melanarius was identified as the main scavenger. Comparison of the retention time for dead slugs in the field, with the detection period for decaying slug material in the guts of the predators, showed that PCR-based techniques are not able to distinguish between predated and scavenged food items. This could potentially lead to overestimation of the impact of predation on slugs (and other prey) by carabids. Possible implications of facultative scavenging by invertebrate predators for biocontrol and food-web research are discussed.

The drive towards a more sustainable and integrated approach to pest management has engendered a renewed interest in conservation biological control, the role of natural enemy communities and their interactions with prey. Monoclonal antibodies have provided significant advances in enhancing our knowledge of trophic interactions and can be employed to help quantify predation on target species. The tetragnathid spider Pachygnatha degeeri Sundevall was collected from fields of winter wheat in the UK and assayed by ELISA for aphid proteins. It was demonstrated that this spider did not simply consume greater quantities of aphids because it was bigger. In addition, P. degeeri contained significantly greater concentrations of aphid in their guts than other spiders, showing that aphids comprised a greater proportion of their diet. Although P. degeeri constituted only 6% of the spider population numerically, females and males respectively contained 16% and 37% of total aphid proteins within all spiders screened, significantly more than their density would predict. These spiders also preyed upon aphids at a disproportionately high rate in June, during the aphid establishment phase, theoretically the best time for limiting growth in the aphid population. Although less abundant than other generalist predators, the capability of these hunting spiders to consume large numbers of aphids highlights them as a more significant component of the predator complex than had previously been realized. Limitation of aphid numbers early in the year by generalist predators provides more time for the specialist aphid predators and parasitoids to move in.