Molecular analysis of predation: a review of best practice for DNA-based approaches

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.     

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.     

Ant predation on red oak borer confirmed by field observation and molecular gut-content analysis

1 Populations of an indigenous longhorn beetle, the red oak borer Enaphalodes rufulus (Haldeman) (Coleoptera: Cerambycidae), recently reached epidemic levels in the Ozark National Forests of Arkansas and Missouri, resulting in extensive tree mortality.
2 The factors regulating E. rufulus populations are largely unknown. Ants appear to be the most abundant potential predators in the Ozarks and have been shown to play a role in regulating populations of other forest insects.
3 The main objective of the present study was to determine whether ants are predators of early E. rufulus life stages by direct observation of E. rufulus eggs artificially placed on tree trunks and by development of molecular tools to detect E. rufulus DNA within ant gut contents.
4 Three hundred and eighty E. rufulus eggs were applied to ten northern red oaks. Ants ate or carried away 72% of the eggs within 1 h. Camponotus pennsylvanicus (De Geer) and Aphaenogaster tennesseensis (Mayr) were identified as the two primary ant species observed in the study.
5 A portion of the mitochondrial 16S rRNA gene of E. rufulus was sequenced and polymerase chain reaction primers were developed to detect E. rufulus DNA in the guts of C. pennsylvanicus . Enaphalodes rufulus DNA persisted in ant gut contents for at least 24 h after ingestion under laboratory conditions, and E. rufulus DNA was detected in field-collected ant populations, suggesting the natural occurrence of ant predation on this insect.     

Predation on exotic zebra mussels by native fishes: effects on predator and prey

SUMMARY 1. Exotic zebra mussels, Dreissena polymorpha, occur in southern U.S. waterways in high densities, but little is known about the interaction between native fish predators and zebra mussels. Previous studies have suggested that exotic zebra mussels are low profitability prey items and native vertebrate predators are unlikely to reduce zebra mussel densities. We tested these hypotheses by observing prey use of fishes, determining energy content of primary prey species of fishes, and conducting predator exclusion experiments in Lake Dardanelle, Arkansas. 2. Zebra mussels were the primary prey eaten by 52.9% of blue catfish, Ictalurus furcatus; 48.2% of freshwater drum, Aplodinotus grunniens; and 100% of adult redear sunfish, Lepomis microlophus. Blue catfish showed distinct seasonal prey shifts, feeding on zebra mussels in summer and shad, Dorosoma spp., during winter. Energy content (joules g⁻¹) of blue catfish prey (threadfin shad, Dorosoma petenense; gizzard shad, D. cepedianum; zebra mussels; and asiatic clams, Corbicula fluminea) showed a significant species by season interaction, but shad were always significantly greater in energy content than bivalves examined as either ash-free dry mass or whole organism dry mass. Fish predators significantly reduced densities of large zebra mussels (>5 mm length) colonising clay tiles in the summers of 1997 and 1998, but predation effects on small zebra mussels (≤5 mm length) were less clear. 3. Freshwater drum and redear sunfish process bivalve prey by crushing shells and obtain low amounts of higher-energy food (only the flesh), whereas blue catfish lack a shell-crushing apparatus and ingest large amounts of low-energy food per unit time (bivalves with their shells). Blue catfish appeared to select the abundant zebra mussel over the more energetically rich shad during summer, then shifted to shad during winter when shad experienced temperature-dependent stress and mortality. Native fish predators can suppress adult zebra mussel colonisation, but are ultimately unlikely to limit population density because of zebra mussel reproductive potential.     

Tracking the role of alternative prey in soybean aphid predation by Orius insidiosus: a molecular approach

The soybean aphid, Aphis glycines (Hemiptera: Aphididae), is a pest of soybeans in Asia, and in recent years has caused extensive damage to soybeans in North America. Within these agroecosystems, generalist predators form an important component of the assemblage of natural enemies, and can exert significant pressure on prey populations. These food webs are complex and molecular gut-content analyses offer nondisruptive approaches for examining trophic linkages in the field. We describe the development of a molecular detection system to examine the feeding behaviour of Orius insidiosus (Hemiptera: Anthocoridae) upon soybean aphids, an alternative prey item, Neohydatothrips variabilis (Thysanoptera: Thripidae), and an intraguild prey species, Harmonia axyridis (Coleoptera: Coccinellidae). Specific primer pairs were designed to target prey and were used to examine key trophic connections within this soybean food web. In total, 32% of O. insidiosus were found to have preyed upon A. glycines, but disproportionately high consumption occurred early in the season, when aphid densities were low. The intensity of early season predation indicates that O. insidiosus are important biological control agents of A. glycines, although data suggest that N. variabilis constitute a significant proportion of the diet of these generalist predators. No Orius were found to contain DNA of H. axyridis, suggesting intraguild predation upon these important late-season predators during 2005 was low. In their entirety, these results implicate O. insidiosus as a valuable natural enemy of A. glycines in this soybean agroecosystem.     

A new method for immunologically marking prey and its use in predation studies

We introduce a new method for immunologically examining predator gut contents. It differs from previously described gut content analyses because it does not require the development of prey-specific antibody probes. Instead, insect prey were marked with a readily available antigen, rabbit immunoglobulin G (IgG). We then assayed predators that had fed on IgG labeled prey with an enzyme-linked immunosorbent assay (ELISA) using goat anti-rabbit IgG. Of the predator species that fed on the IgG labeled prey, 98.8% of those with chewing mouthparts scored positive for IgG 1 h after feeding. Our prey-labeling ELISA was less efficient for detecting IgG prey remains in predators with piercing/sucking mouthparts. Only 29.5% of these individuals scored positive for rabbit IgG in their guts 1 h after feeding. An additional study was conducted to measure the retention time of IgG-labeled prey in the guts of two species of predators with chewing mouthparts. Results from this experiment showed that the retention time varied depending on the predator and prey species examined. Results from these studies indicate that this marking technique could have widespread use for analyzing the gut contents of predators with chewing mouthparts, but it has limited application for those predators with piercing/sucking mouthparts. This article presents the results of research only. Mention of a proprietary product does not constitute an endorsement or recommendation for its use by the USDA.     

Prey DNA detection success following digestion by intraguild predators: influence of prey and predator species

Intraguild predation (IGP) has been increasingly recognized as an important interaction in ecological systems over the past two decades, and remarkable insights have been gained into its nature and prevalence. We have developed a technique using molecular gut-content analysis to compare the rate of IGP between closely related species of coccinellid beetles (lady beetles or ladybirds), which had been previously known to prey upon one another. We first developed PCR primers for each of four lady beetle species: Harmonia axyridis, Coccinella septempunctata, Coleomegilla maculata and Propylea quatuordecimpunctata. We next determined the prey DNA detection success over time (DS(50) ) for each combination of interacting species following a meal. We found that DS(50) values varied greatly between predator-prey combinations, ranging from 5.2 to 19.3 h. As a result, general patterns of detection times based upon predator or prey species alone are not discernable. We used the DS(50) values to correct field data to demonstrate the importance of compensation for detection times that are specific to particular predator-prey combinations.     

Predator diversity and the functioning of ecosystems: the role of intraguild predation in dampening trophic cascades

Single trophic‐level studies of the relationship between biodiversity and ecosystem functioning highlight the importance of mechanisms such as resource partitioning, facilitation, and sampling effect. In a multi‐trophic context, trophic interactions such as intraguild predation may also be an important mediator of this relationship. Using a salt‐marsh food web, we investigated the interactive effects of predator species richness (one to three species) and trophic composition (strict predators, intraguild predators, or a mixture of the two) on ecosystem functions such as prey suppression and primary production via trophic cascades. We found that the trophic composition of the predator assemblage determined the impact of increasing predator species richness on the occurrence of trophic cascades. In addition, increasing the proportion of intraguild predator species present diminished herbivore suppression and reduced primary productivity. Therefore, trophic composition of the predator assemblage can play an important role in determining the nature of the relationship between predator diversity and ecosystem function.     

Differential predation by the generalist predator Orius insidiosus on congeneric species of thrips that vary in size and behavior

We investigated interactions between the generalist predator Orius insidiosus (Say) (Heteroptera: Anthocoridae) and two species of thrips prey, Frankliniella bispinosa (Morgan) and Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), and interspecific differences in morphology and behavior between these prey species that could contribute to differences in predation by O. insidiosus. Frankliniella occidentalis is significantly larger than F. bispinosa. Frankliniella bispinosa has greater mobility compared with F. occidentalis. When O. insidiosus was offered either F. bispinosa or F. occidentalis as prey in single species trials, there were no significant differences in the number of prey captured. However, O. insidiosus had significantly more encounters with F. bispinosa than with F. occidentalis. In arenas with equal numbers of both species, O. insidiosus encountered and captured F. occidentalis more than F. bispinosa. In large arenas with two pepper plants (Capsicum annuum L.), O. insidiosus preyed on more F. occidentalis than on F. bispinosa. These results indicate that O. insidiosus can prey on both thrips species, but that it preferentially captures F. occidentalis. The greater locomotion and movement of F. bispinosa, perhaps combined with its smaller size, allow it to evade predation by O. insidiosus better than F. occidentalis. Consequently, the observed preference of O. insidiosus for F. occidentalis is not exclusively a function of active selection by the predator but also could arise from inherent differences among prey. We propose this differential predation as a mechanism contributing to observed differences in the temporal dynamics of these species in pepper fields.     

Molecular identification of predation by carabid beetles on exotic and native slugs in a strawberry agroecosystem

Generalist predators have the capacity to exert significant pressure on prey populations. However, integrating them into biological control programs relies on a detailed understanding of their foraging behavior and the levels of trophic connectedness with pest species. Carabid beetles are important predators of slugs, pests of agricultural, floricultural and horticultural crops worldwide, but these interactions have been rarely studied outside the Western Palearctic ecozone. Diagnostic molecular gut-content analysis was used to examine the strength of trophic pathways between a community of carabid beetles and two slug species, the exotic Deroceras reticulatum and native Deroceras laeve, in strawberry agroecosystems. Strawberries were grown according to standard horticultural practices for central Kentucky, following traditional bare ground planting or with the addition of detrital subsidies, to quantify the impact of habitat management on the abundance of pests and the strength of these trophic pathways. Following laboratory characterization of species-specific molecular markers targeting both Deroceras species, carabid beetles collected from a strawberry agroecosystem were screened for slug DNA. Field collections revealed important food web pathways existed between Harpalus pensylvanicus and D. reticulatum, with 7.2% screening positive for these prey yet none screening positive for D. laeve. In contrast, Chlaenius tricolor was found to feed on both slug species in the field, with 16% screening positive for both Deroceras. Despite below normal rainfall limiting slug densities in the field, the results presented here reveal the potential importance of carabid beetles in slug population dynamics in the Nearctic.     

Herbivory on a chemically defended plant as a predation deterrent in Hyalella azteca

SUMMARY 1. We investigated whether a population of the freshwater amphipod, Hyalella azteca , which consumed plants with defensive secondary compounds, reduced predation as reported for terrestrial and marine systems.
2. Field observations in Montezuma Well, U.S.A., indicated a strong association between the emergent macrophyte, Berula erecta and H . azteca . We hypothesised that this geographically isolated population of sedentary amphipods was able to consume roots of the chemically defended B . erecta as a deterrent against predation. Berula erecta is in the family Apiaceae, which commonly produce coumarins that deter herbivory in terrestrial systems.
3.  Hyalella azteca consumed roots of B . erecta at a significantly greater rate than alternative macrophyte substrata in Montezuma Well. Additionally, H . azteca moulted at a significantly higher rate when consuming B . erecta compared with a diet of periphyton.
4. Two insect predators ( Telebasis salva and Belostoma bakeri ) with different feeding strategies were used to assess the effects of a B . erecta diet on predation rates in the laboratory and in Montezuma Well. Hyalella azteca was preyed on at a significantly lower rate by both predators when given a strict diet of B . erecta roots compared with a diet of periphyton.
5. This is the first experimental evidence that predation on a freshwater herbivore, H . azteca , was reduced when it consumed a chemically defended plant.     

Predation of a cave fish (Poecilia mexicana, Poeciliidae) by a giant water-bug (Belostoma, Belostomatidae) in a Mexican sulphur cave

Abstract.  1. Caves are often assumed to be predator-free environments for cave fishes. This has been proposed to be a potential benefit of colonising these otherwise harsh environments. In order to test this hypothesis, the predator–prey interaction of a belostomatid (predator) and a cave fish (prey) occurring in the Cueva del Azufre (Tabasco, Mexico) was investigated with two separate experiments.
2. In one experiment, individual Belostoma were given a chance to prey on a cave fish, the cave form of the Atlantic molly ( Poecilia mexicana ), to estimate feeding rates and size-specific prey preferences of the predator. In the other experiment, population density of Belostoma was estimated using a mark–recapture analysis in one of the cave chambers.
3. Belostomatids were found to heavily prey on cave mollies and to exhibit a prey preference for large fish. The mark–recapture analysis revealed a high population density of the heteropterans in the cave.
4. The absence of predators in caves is not a general habitat feature for cave fishes. None the less predation regimes differ strikingly between epigean and hypogean habitats. The prey preference of Belostoma indicates that cave-dwelling P. mexicana experience size-specific predation pressure comparable with surface populations, which may have implications for life-history evolution in this cave fish.     

Collembola as alternative prey sustaining spiders in arable ecosystems: prey detection within predators using molecular markers

Collembola comprise a major source of alternative prey to linyphiid spiders in arable fields, helping to sustain and retain these predators as aphid control agents within the crop. Polymerase chain reaction primers were developed for the amplification, from spider gut samples, of DNA from three of the most abundant species of Collembola in wheat crops in Europe, namely Isotoma anglicana, Lepidocyrtus cyaneus and Entomobrya multifasciata. The primers amplified fragments of the mitochondrial cytochrome oxidase subunit I (COI) gene and were designed following alignment of comparable sequences for a range of predator and prey species. Each of the primer pairs proved to be species-specific to a Collembola species, amplifying DNA fragments from 211 to 276 base pairs in length. Following consumption of a single collembolan, prey DNA was detectable in 100% of spiders after 24 h of digestion. We report the first use of DNA-based techniques to detect predation by arthropods on natural populations of prey in the field. All three species of Collembola were consumed by the spiders. By comparing the ratios of the Collembola species in the field with the numbers of spiders that gave positive results for each of those species, it was possible to demonstrate that the spiders were exercising prey choice. Overall, a single target species of Collembola was eaten by 48% of spiders while a further 16% of spiders contained DNA from two different species of Collembola. Preference was particularly evident for I. anglicana, the species most frequently found in spider guts yet the least numerous of the three target species in the field.     

Molecular diagnostics reveal spiders that exploit prey vibrational signals used in sexual communication

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.     

Population- and ecosystem-level effects of predation on microbial-feeding nematodes

We studied the role of nematode predation in the functioning of detrital food webs assembled in microcosms. The microcosms contained defaunated humus and litter materials, a diverse microbial community with bacteria, fungi and protozoa, and a birch (Betula pendula) seedling infected with mycorrhizal fungi. Different levels of top-down control upon microbivorous nematodes were set up by assembling food webs either without predators, or in combinations with a specialist and a non-specialist predatory mite (Mesostigmata). The nematode community was composed of either (1) three species of bacterivorous, or (2) three species of fungivorous nematodes or (3) both groups together. After two growing periods for the birch (38 weeks), the microcosms were destructively sampled for animal and microbial biomasses, concentration of mineral N in the soil, plant biomass and plant N concentration. The specialist predator reduced biomasses of both bacterial- and fungal-feeding nematodes by more than 50%, whereas the non-specialist predator weakly increased the biomass of fungivorous nematodes. Thus, under high predation pressure, the biomass of microbivores changed as predicted by trophic dynamic models assuming strong top-down control and uniformly behaving trophic levels. Despite this, microbial biomass was unaffected by the predators. However, microbial respiration increased slightly in the presence of predators. Assuming that microbial respiration correlates with microbial productivity, the increase in microbial respiration indicates a cascading productivity regulation. The composition of the microbivore community had only a minor effect on the outcome of the top-down control on microbes. The >50% reduction in nematode biomass and respiration coincided with <16% increase in microbial respiration and did not affect microbial biomass. Presence of the specialist predator slightly reduced soil NH⁺ ₄ concentration in communities with fungivore nematodes but plant growth and N uptake remained unchanged. Thus, the structure of the community only weakly controlled nutrient mineralisation. Received: 18 May 1998 / Accepted: 3 May 1999     

A pragmatic approach to the analysis of diets of generalist predators: the use of next‐generation sequencing with no blocking probes

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.     

Compared with conventional PCR assay,qPCR assay greatly improves the detection efficiency of predation

Studies of predation can contribute greatly to understanding predator–prey relationships and can also provide integral knowledge concerning food webs and multi‐trophic level interactions. Both conventional polymerase chain reaction (cPCR) and quantitative PCR (qPCR) have been employed to detect predation in the field because of their sensitivity and reproducibility. However, to date, few studies have been used to comprehensively demonstrate which method is more sensitive and reproducible in studies of predation. We used a Drosophila melanogaster‐specific DNA fragment (99 bp) to construct a tenfold gradient dilution of standards. Additionally, we obtained DNA samples from Pardosa pseudoannulata individuals that fed on D. melanogaster at various time since feeding. Finally, we compared the sensitivity and reproducibility between cPCR and qPCR assays for detecting DNA samples from feeding trials and standards. The results showed that the cPCR and qPCR assays could detect as few as 1.62 × 10³ and 1.62 × 10¹ copies of the target DNA fragment, respectively. The cPCR assay could detect as few as 48 hr post‐feeding of the target DNA fragment. But the qPCR assay showed that all spiders were positive after consuming prey at various time intervals (0, 24, 48, 72, and 96 hr). A smaller proportion of the technical replicates were positive using cPCR, and some bands on the agarose gel were absent or gray, while some were white and bright for the same DNA samples after amplification by cPCR. By contrast, a larger proportion of the technical replicates were positive using qPCR and the coefficients of variation of the Ct value for the three technical replicates of each DNA sample were less than 5%. These data showed that qPCR was more sensitive and highly reproducible in detecting such degraded DNA from predator's gut. The present study provides an example of the use of cPCR and qPCR to detect the target DNA fragment of prey remains in predator's gut.     

Patterns of exclusion in an intraguild predator-prey system depend on initial conditions

1. When intraguild (IG)-prey are superior to IG-predators in competing for a shared resource, theory predicts coexistence of the IG-prey or the IG-predator with the resource depending on the productivity level: (a) resource and IG prey coexist when productivity is low; (b) IG-predator and resource coexist at high productivity; (c) if IG-prey and IG-predators can coexist, it is only at intermediate productivity levels. 2. We tested the existence of productivity-dependent regions of coexistence using an experimental system of two predatory mites and a shared food source (pollen). 3. At high levels of pollen supply (i.e. high productivity), the IG-predator excluded the IG-prey in most, but not all, cases. The same pattern of exclusion was observed at low productivity, at which the IG-prey was expected to exclude the IG-predator. Therefore, species composition could not be predicted by productivity levels. Instead, our results show that initial conditions affected strongly the outcome of the interaction. 4. We emphasize the need for theory on IG-predation that takes the effects of stage structure, initial conditions and transient dynamics into account.