Multiplex reactions for the molecular detection of predation on pest and nonpest invertebrates in agroecosystems

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.     

The significance of facultative scavenging in generalist predator nutrition: detecting decayed prey in the guts of predators using PCR

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.     

Detection of secondary predation by PCR analyses of the gut contents of invertebrate generalist 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.     

Rapid screening of invertebrate predators for multiple prey DNA targets

DNA-based techniques are providing valuable new approaches to tracking predator-prey interactions. The gut contents of invertebrate predators can be analysed using species-specific primers to amplify prey DNA to confirm trophic links. The problem is that each predator needs to be analysed with primers for the tens of potential prey available at a field site, even though the mean number of species detected in each gut may be as few as one or two. Conducting all these PCRs (polymerase chain reactions) is a lengthy process, and effectively precludes the analysis of the hundreds of predators that might be required for a meaningful ecological study. We report a rapid, more sensitive and practical approach. Multiplex PCRs, incorporating fluorescent markers, were found to be effective at amplifying degraded DNA from predators' guts and could amplify mitochondrial DNA fragments from 10+ species simultaneously without 'drop outs'. The combined PCR products were then separated by size on polyacrylamide gels on an ABI377 sequencer. New primers to detect the remains of aphids, earthworms, weevils and molluscs in the guts of carabid predators were developed and characterized. The multiplex-sequencer approach was then applied to field-caught beetles, some of which contained DNA from as many as four different prey at once. The main prey detected in the beetles proved to be earthworms and molluscs, although aphids and weevils were also consumed. The potential of this system for use in food-web research is discussed.     

应用TGGE技术分析人肠道中双歧杆菌的组成

用温度梯度凝胶电泳(TGGE)技术结合16SrDNA克隆、测序,对健康人肠道中双歧杆菌的组成进行了分析。10例健康人肠道双歧杆菌的TGGE分析显示:人肠道内双歧杆菌的组成具有宿主特异性,不同人肠道双歧杆菌种的多样性和种类不同。通过对一健康儿童肠道双歧杆菌属特异性PCR扩增产物的测序及TGGE电泳行为的分析发现,该个体双歧杆菌TGGE图谱中的条带分别代表Bifidobacteriumbifidum、B.infantis、B.longum、B.adolescentis、B.pseudocatenulatum等种和一新种,其中B.pseudocatenulatum(假小链双歧杆菌)是大多数个体共有且较优势的种类。用传统培养方法只检出B.pseudocatenulatum和B.longum两种。基于双歧杆菌属特异性PCR基础上的TGGE方法结合16SrDNA克隆文库分析可较灵敏、直观地反映人肠道中双歧杆菌的组成。     

Biodiversity vs. biocontrol: positive and negative effects of alternative prey on control of slugs by carabid beetles

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.     

Feeding mode and prey detectability half-lives in molecular gut-content analysis: an example with two predators of the Colorado potato beetle

The time during which prey remains are detectable in the gut of a predator is an important consideration in the interpretation of molecular gut-content data, because predators with longer detectability times may appear on the basis of unweighted data to be disproportionately important agents of prey population suppression. The rate of decay in detectability, typically expressed as the half-life, depends on many variables; one that has not been explicitly examined is the manner in which the predator processes prey items. The influence of differences in feeding mode and digestive physiology on the half-life of DNA for a single prey species, the Colorado potato beetle Leptinotarsa decemlineata (Say), is examined in two predators that differ dramatically in these attributes: the pink ladybeetle, Coleomegilla maculata (DeGeer), which feeds by chewing and then ingesting the macerated material into the gut for digestion; and the spined soldier bug, Podisus maculiventris (Say), which physically and enzymatically processes the prey extra-orally before ingestion and further digestion in the gut. In order to standardize the amount of DNA consumed per predator, a single L. decemlineata egg was used as the prey item; all predators were third instars. The PCR assay yields estimated prey DNA half-lives, for animals maintained under field temperatures, of 7.0 h in C. maculata and 50.9 h in P. maculiventris. The difference in the prey DNA half-lives from these two predators underscores the need to determine detectabilities from assemblages of predators differing in feeding mode and digestive physiology, in order to weight positives properly, and hence determine the predators' relative impacts on prey population suppression.     

Molecular profiling of Bacteroides spp. in human feces by PCR-temperature gradient gel electrophoresis

A group-specific PCR-based temperature gradient gel electrophoresis (TGGE) method was developed to study the population composition of genus Bacteroides in human gut. Highly reproducible and well-separated bands in TGGE fingerprints of ten unrelated human fecal samples showed complex and host-specific Bacteroides species composition. Dynamic monitoring over 22 months of samples from one healthy 10-year-old boy indicated a relatively stable population profile of Bacteroides. The species identity of each band in TGGE gel of this boy was also resolved via comigration analysis of sequenced inserts in a Bacteroides group-specific clone library. This work provides a rapid and effective technique for analyzing the species composition of Bacteroides in human gut.     

The effects of temperature on detection of prey DNA in two species of carabid beetle

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.     

Anurans as prey: an exploratory analysis and size relationships between predators and their prey

The vertebrate predators of post-metamorphic anurans were quantified and the predator–prey relationship was investigated by analysing the relative size of invertebrate predators and anurans. More than 100 vertebrate predators were identified (in more than 200 reports) and classified as opportunistic, convenience, temporary specialized and specialized predators. Invertebrate predators were classified as solitary non-venomous, venomous and social foragers according to 333 reviewed reports. Each of these categories of invertebrate predators was compared with the relative size of the anurans, showing an increase in the relative size of the prey when predators used special predatory tactics. The number of species and the number of families of anurans that were preyed upon did not vary with the size of the predator, suggesting that prey selection was not arbitrary and that energetic constraints must be involved in this choice. The relatively low predation pressure upon brachycephalids was related to the presence of some defensive strategies of its species. This compounding review can be used as the foundation for future advances in vertebrate predator–prey interactions.     

Group-Specific Multiplex PCR Detection Systems for the Identification of Flying Insect Prey

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.     

A tale of two fluids: does storing specimens together in liquid preservative cause DNA cross‐contamination in molecular gut‐content studies?

The study of food webs and trophic interactions increasingly relies on PCR‐based molecular gut‐content analysis. However, this technique may be prone to error from contamination of minute quantities of DNA; i.e., simply storing specimens together in a liquid medium may lead to cross‐contamination. In this study, we used PCR to determine the contamination rate when (1) specimens were stored together in 95% ethanol for various time periods, and (2) predators fall into ethylene glycol‐filled pitfall traps where the dying predator may inadvertently consume prey DNA‐contaminated liquid. We designed experiments and PCR primers to quantify the risk of contamination for both situations and found no contamination by storing specimens together in 95% ethanol. Furthermore, zero predators contained prey DNA in their gut contents from imbibing prey DNA‐contaminated ethylene glycol. These results support the use of mass sampling techniques, like wet pitfall traps, for molecular gut‐content analysis.     

Molecular gut-content analysis of a predator assemblage reveals the effect of habitat manipulation on biological control in the field

Habitat manipulation in agroecosystems can influence predator–prey interactions. In this study, we collected foliar predators from field potato plots with different mulch treatments and assayed them for DNA of the target prey, Leptinotarsa decemlineata (Say), using species-specific primers. Concurrently, L. decemlineata larval abundance and plant damage were recorded from the same plots. Predator species abundance and diversity were not influenced by habitat manipulation, while prey density was highest in plots without mulch. Gut-content analysis revealed that the highest incidence of predators positive for L. decemlineata DNA was in plots without mulch, where target prey abundance was highest. Therefore, the lower prey abundance in mulched plots was not due to predation. The most abundant species in the predator assemblage was Coleomegilla maculata, which had the lowest proportion of L. decemlineata DNA in the gut. Podisus maculiventris, Perillus bioculatus, and Lebia grandis were less abundant but had a higher incidence of target prey DNA in the gut. DNA detectability half-lives were used to adjust for inter-specific variation in DNA digestive rates of the four predator species. Using this information to adjust actual number of positives for prey DNA, we compared proportions positive for L. decemlineata and found that P. maculiventris is the most effective predator species in the complex.     

Do earthworms help to sustain the slug predator Pterostichus melanarius (Coleoptera: Carabidae) within crops? Investigations using monoclonal antibodies

Earthworms provide a major potential source of alternative food for polyphagous predators, such as carabid beetles, that are natural enemies of slugs, aphids and other agricultural pests. Non-pest prey may foster larger numbers of natural enemies, which then help to control pests, or alternatively may help to divert the predators away from pest control. An earthworm-specific monoclonal antibody was developed to study carabid-earthworm interactions in the field and assess the role of earthworms as alternative prey. The antibody could identify as little at 7 ng of earthworm protein in an ELISA, and could detect earthworm remains in the foregut of the carabid beetle Pterostichus melanarius for 64 h after consumption. Thirty-six per cent of field-collected beetles contained earthworm remains. Quantities of earthworm proteins in the beetle foreguts were negatively related to total foregut biomass, suggesting that earthworm consumption increased as total prey availability declined. There was also a negative relationship between foregut biomass and beetle numbers, but both quantities and concentrations of earthworm proteins in beetle foreguts were positively related to beetle numbers. This suggests that as beetle activity-density increased, total prey availability declined, or, as prey availability declined, beetles spent more time searching. In these circumstances, beetles fed to a greater extent on earthworms, an acceptable but nonpreferred food item. Earthworms may, therefore, provide an ideal alternative prey for P. melanarius, helping to sustain it when pest numbers are low but allowing it to perform a 'lying-in-wait' strategy, ready to switch back to feeding on pests when they become available.     

Group-specific polymerase chain reaction for DNA-based analysis of species diversity and identity in dietary samples

Unique DNA sequences are present in all species and can be used as biomarkers for the detection of cells from that species. These DNA sequences can most easily be detected using the polymerase chain reaction (PCR), which allows very small quantities of target DNA sequence to be amplified even when the target is mixed with large amounts of nontarget DNA. PCR amplification of DNA markers that are present in a wide range of species has proven very useful for studies of species diversity in environmental samples. The taxonomic range of species to be identified from environmental samples may often need to be restricted to simplify downstream analyses and to ensure that less abundant sequences are amplified. Group-specific PCR primer sets are one means of specifying the range of taxa that produce an amplicon in a PCR. We have developed a range of group-specific PCR primers for studying the prey diversity found in predator stomach contents and scats. These primers, their design and their application to studying prey diversity and identity in predator diet are described.     

Amplified detection, using a monoclonal antibody, of an aphid-specific epitope exposed during digestion in the gut of a predator.

Monoclonal antibodies are invaluable tools for identifying and quantifying prey remains in the fore-guts of predators. However, they must be target-specific, detect an epitope that is well replicated within the prey (to enhance assay sensitivity) and, critically, recognise a site that can resist digestion. A monoclonal antibody is reported that proved to be aphid-specific and capable of detecting, and accurately identifying, as little as 16.5 ng of aphid protein within a heterologous mixture of invertebrate material. The antibody was selected by screening hybridoma lines for antibodies that bound with semi-digested aphid proteins. The antibody detected an epitope that was found, against expectation, to significantly increase in concentration with time (by approx. 50% over 6 h) in the gut of the carabid predator Pterostichus melanarius. The resultant extended antigen detection period and half-life, and the high specificity of this antibody, showed it to be an enhanced tool for studying interactions between aphids and their predators in the field. It was concluded that the antibody was initially generated to a surface epitope on a high molecular weight native protein (> 200 kD). This epitope, however, was then either replicated on internal sites progressively revealed by digestion, or new epitopes became available as the conformation of the protein changed during digestion.     

Density‐dependent and ‐independent effects on the joint use of space by predators and prey in terrestrial arthropod food‐webs

The spatial distribution of predators and their prey is affected by their joint use of space. While the formation of such spatial patterns may be driven by density‐dependent and ‐independent factors our knowledge on the contribution of different land‐use activities on the formation of spatial patterns between predators and prey remains very limited. Agriculture is one of the most prevailing land‐use activities with strong effects on invertebrate densities and structural habitat conditions. Here, we used replicated conventionally and organically managed winter wheat fields to investigate the effects of agricultural land‐use on the spatial patterns of generalist predators and decomposer prey. We then identified the explanatory power of density‐dependent (prey and predator activity density) and density‐independent (vegetation structure) predictors for the observed spatial patterns. Generalist predators were regularly distributed only in conventionally managed fields and this pattern intensified with decreasing Collembola prey availability and increasing spider activity density. Segregation between carabid and spider predators was strongest in fields with lowest wheat plant height, suggesting more intense intraguild interactions in structurally less complex habitats. Collembola were aggregated independent of management and aggregation was strongest in fields with highest Collembola and carabid activity density. Spiders and Collembola prey were associated, but higher aphid densities under conventional management weakened or interrupted this spatial relationship. We conclude that active control of crop plant physiognomy by growth hormones and herbicides in conventionally managed fields promotes predator–predator segregation and that a high availability of aphid prey seems to decouple predator–Collembola prey associations. Our results emphasise the need for a more mechanistic understanding of the effects of land‐use on the formation of spatial patterns and species interactions, especially under scenarios of environmental change and an ongoing loss of biodiversity.     

Development of sequence amplified characterized region (SCAR) markers of helicoverpa armigera: a new polymerase chain reaction-based technique for predator gut analysis

A method is described for the development of DNA markers for detection of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in predator gut analysis, based on sequence characterized amplified regions (SCARs) derived from a randomly amplified polymorphic DNA (RAPD) band. A 1200-bp DNA fragment of H. armigera, absent in the predator band pattern and in other closely related prey species, was identified by RAPD analysis. This fragment was cloned and its extremes sequenced to design extended strand-specific 20-mer oligonucleotide primers. Three pairs of SCAR primers, which amplified three different DNA fragments, were used to study the effect of fragment length on detection of prey in the predator gut. Using the pair of primers that amplified the longest fragment of H. armigera DNA, a single band of 1100 bp was obtained, but its detection was not possible in the predator gut. Detection of the ingested prey was possible with the other two pairs of SCAR primers, obtaining bands of 600 and 254 bp, respectively. Detection of H. armigera DNA in the gut of the predator Dicyphus tamaninii was evaluated immediately after ingestion (t = 0) and after 4 h. Detection of H. armigera DNA after 4 h was only possible using the pair of primers that amplified the shortest fragment (254 bp). The test for specificity, using these last pair of primers, showed that H. armigera was the only species detected. The detection threshold was defined at a 1:8192 dilution of a H. armigera whole egg in all samples.     

The impact of a sit-and-wait predator: separating consumption and prey emigration

Reviews of the impact of invertebrate predators in enclosure/exclosure experiments suggest that much of the apparent depletion of prey is due to prey emigration induced by the predators. However, these generalisations derive mainly from studies of invertebrate predators that are predominantly active searchers (usually stoneflies) and of prey with strong avoidance responses (mainly mayflies).
We examined the impact of a large sit-and-wait predator, the nymph of the dragonfly Cordulegaster boltonii , which has recently invaded Broadstone Stream as a new top predator. Field enclosure/exclosure experiments were conducted to assess the impact of the invader on the benthos. Depletion of prey varied seasonally and among taxa, and was highest when prey density and encounter rates were high. Mobile prey, although least likely to show a statistically significant response because of high exchange rates, were those most strongly depleted.
Experimental channels were used to separate the relative contribution of consumption and emigration to total impact for the two most depleted prey species. Depletion of prey was due solely to consumption and predators did not induce emigration. We therefore urge caution in making generalisations about the impacts of invertebrate predators, since sit-and-wait and searching predators potentially have very different impacts.     

PCR enrichment techniques to identify the diet of predators

The increasing sensitivity of PCR has meant that in the last two decades PCR has emerged as a major tool in diet studies, enabling us to refine our understanding of trophic links and to elucidate the diets of predators whose prey is as yet uncharacterized. The achievements and methods of PCR-based diet studies have been reviewed several times, but here we review an important development in the field: the use of PCR enrichment techniques to promote the amplification of prey DNA over that of the predator. We first discuss the success of using group-specific primers either in parallel single reactions or in multiplex reactions. We then concentrate on the more recent use of PCR enrichment techniques such as restriction enzyme digests, peptide nucleic acid clamping, DNA blocking and laser capture microdissection. We also survey the vast literature on enrichment techniques in clinical biology, to ascertain the pitfalls of enrichment techniques and what refinements have yielded some highly sensitive methods. We find that while there are several new approaches to enrichment, peptide nucleic acid clamping and DNA blocking are generally sufficient techniques for the characterization of diets of predators and highlight the most important considerations of the approach.