Evaluation of temperature gradient gel electrophoresis for the analysis of prey DNA within the guts of invertebrate predators

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.     

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.     

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.     

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.     

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.     

Ranking common predators of Bemisia tabaci in Georgia (USA) agricultural landscapes with diagnostic PCR: implications of primer specific post-feeding detection time

Developing a successful biological control program relies on understanding predator–prey interactions in agroecosystem field settings. Among several methods used, molecular gut content analysis (MGCA) has become a popular method to measure predator contributions to pest control services. Once MGCA is applied to diagnose predator–prey interactions, the DNA detectability half-life is often applied to adjust for differences in prey digestion time among predators. Although MGCA best practices are well established, with many primers available, further work is needed to rank among published primers for MGCA. Using a combination of laboratory feeding trials and application of diagnostic MGCA to field collected predators, we investigated Bemisia tabaci post-feeding detection times in three dominant predator functional groups (chewing, piercing/sucking, and spiders). This was based on three published B. tabaci-specific primers. These data reveal that primer choice generated significantly different B. tabaci DNA half-lives in predator gut content. The primers with longer half-life resulted in higher field predation frequency estimation. Our field data using the primer with the longest half-life suggest several abundant predators, including Hippodamia convergens, Geocoris punctipes, Orius spp., Thomisidae spider, and fire ants (Solenopsis invicta), are actively feeding on B. tabaci in cotton fields. Orius spp. and fire ants were the most abundant predator species in our study area and contributed the most to B. tabaci control. Our results suggest that primers can be classified based on their specific DNA half-lives and can be used to address different ecological questions such as how to study time-specific predation detection (nocturnal or diurnal).

     

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.     

Additive effects of predator diversity on pest control caused by few interactions among predator species

1. Studies of the impact of predator diversity on biological pest control have shown idiosyncratic results. This is often assumed to be as a result of differences among systems in the importance of predator–predator interactions such as facilitation and intraguild predation. The frequency of such interactions may be altered by prey availability and structural complexity. A direct assessment of interactions among predators is needed for a better understanding of the mechanisms affecting prey abundance by complex predator communities. 2. In a field cage experiment, the effect of increased predator diversity (single species vs. three‐species assemblage) and the presence of weeds (providing structural complexity) on the biological control of cereal aphids were tested and the mechanisms involved were investigated using molecular gut content analysis. 3. The impact of the three‐predator species assemblages of aphid populations was found to be similar to those of the single‐predator species treatments, and the presence or absence of weeds did not alter the patterns observed. This suggests that both predator facilitation and intraguild predation were absent or weak in this system, or that these interactions had counteracting effects on prey suppression. Molecular gut content analysis of predators provided little evidence for the latter hypothesis: predator facilitation was not detected and intraguild predation occurred at a low frequency. 4. The present study suggests additive effects of predators and, therefore, that predator diversity per se neither strengthens nor weakens the biological control of aphids in this system.     

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.     

Seasonally varying marine influences on the coastal ecosystem detected through molecular gut analysis

Terrestrial predators on marine shores benefit from the inflow of organisms and matter from the marine ecosystem, often causing very high predator densities and indirectly affecting the abundance of other prey species on shores. This indirect effect may be particularly strong if predators shift diets between seasons. We therefore quantified the seasonal variation in diet of two wolf spider species that dominate the shoreline predator community, using molecular gut content analyses with general primers to detect the full prey range. Across the season, spider diets changed, with predominantly terrestrial prey from May until July and predominantly marine prey (mainly chironomids) from August until October. This pattern coincided with a change in the spider age and size structure, and prey abundance data and resource selection analyses suggest that the higher consumption of chironomids during autumn is due to an ontogenetic diet shift rather than to variation in prey abundance. The analyses suggested that small dipterans with a weak flight capacity, such as Chironomidae, Sphaeroceridae, Scatopsidae and Ephydridae, were overrepresented in the gut of small juvenile spiders during autumn, whereas larger, more robust prey, such as Lepidoptera, Anthomyidae and Dolichopodidae, were overrepresented in the diet of adult spiders during spring. The effect of the inflow may be that the survival and growth of juvenile spiders is higher in areas with high chironomid abundances, leading to higher densities of adult spiders and higher predation rates on the terrestrial prey next spring.     

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.     

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.     

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.     

Introduced species provide a novel temporal resource that facilitates native predator population growth

Non-native species are recognized as important components of change to food web structure. Non-native prey may increase native predator populations by providing an additional food source and simultaneously decrease native prey populations by outcompeting them for a limited resource. This pattern of apparent competition may be important for plants and sessile marine invertebrate suspension feeders as they often compete for space and their immobile state make them readily accessible to predators. Reported studies on apparent competition have rarely been examined in biological invasions and no study has linked seasonal patterns of native and non-native prey abundance to increasing native predator populations. Here, we evaluate the effects of non-native colonial ascidians (Diplosoma listerianum and Didemnum vexillum) on population growth of a native predator (bloodstar, Henricia sanguinolenta) and native sponges through long-term surveys of abundance, prey choice and growth experiments. We show non-native species facilitate native predator population growth by providing a novel temporal resource that prevents loss of predator biomass when its native prey species are rare. We expect that by incorporating native and non-native prey seasonal abundance patterns, ecologists will gain a more comprehensive understanding of the mechanisms underlying the effects of non-native prey species on native predator and prey population dynamics.     

Two common invertebrate predators show varying predation responses to different types of sentinel prey

Sentinel prey (an artificially manipulated patch of prey) are widely used to assess the level of predation provided by natural enemies in agricultural systems. Whilst a number of different methodologies are currently in use, little is known about how arthropod predators respond to artificially manipulated sentinel prey in comparison with predation on free‐living prey populations. We assessed how attack rates on immobilized (aphids stuck to cards) and artificial (plasticine lepidopteran larvae mimics) sentinel prey differed to predation on free‐moving live prey (aphids). Predation was assessed in response to density of the common invertebrate predators, a foliar‐active ladybird Harmonia axyridis (Coleoptera: Coccinellidae), and a ground‐active beetle Pterostichus madidus (Coleoptera: Carabidae). Significant increases in attack rates were found for the immobilized and artificial prey between the low and high predator density treatments. However, an increased predator density did not significantly reduce numbers of free‐living live aphids included in the mesocosms in addition to the alternate prey. We also found no signs of predation on the artificial prey by the predator H. axyridis. These findings suggest that if our assessment of predation had been based solely on the foliar artificial prey, then no increase in predation would have been found in response to increased predator density. Our results demonstrate that predators differentially respond to sentinel prey items which could affect the level of predation recorded where target pest species are not being used.     

Interspecific differences in antipredator strategies determine the strength of non‐consumptive predator effects on stream detritivores

Animal species differ considerably in their response to predation risks. Interspecific variability in prey behaviour and morphology can alter cascading effects of predators on ecosystem structure and functioning. We tested whether species‐specific morphological defenses may affect responses of leaf litter consuming invertebrate prey to sit‐and‐wait predators, the odonate Cordulegaster boltonii larvae, in aquatic food webs. Partly or completely blocking the predator mouthparts (mandibles and/or extensible labium), thus eliminating consumptive (i.e. lethal) predator effects, we created a gradient of predator‐prey interaction intensities (no predator < predator – no attack < predator – non‐lethal attacks < lethal predator). A field experiment was first used to assess both consumptive and non‐consumptive predator effects on leaf litter decomposition and prey abundances. Laboratory microcosms were then used to examine behavioural responses of armored and non‐armored prey to predation risk and their consequences on litter decomposition. Results show that armored and non‐armored prey responded to both acute (predator – non‐lethal attacks) and chronic (predator – no attack) predation risks. Acute predation risk had stronger effects on litter decomposition, prey feeding rate and prey habitat use than predator presence alone (chronic predation risk). Predator presence induced a reduction in feeding activity (i.e. resource consumption) of both prey types but a shift to predator‐free habitat patches in non‐armored detritivores only. Non‐consumptive predator effects on prey subsequently decreased litter decomposition rate. Species‐specific prey morphological defenses and behaviour should thus be considered when studying non‐consumptive predator effects on prey community structure and ecosystem functioning.     

Effectiveness of Annealing Blocking Primers versus Restriction Enzymes for Characterization of Generalist Diets: Unexpected Prey Revealed in the Gut Contents of Two Coral Reef Fish Species

Characterization of predator-prey interactions is challenging as researchers have to rely on indirect methods that can be costly, biased and too imprecise to elucidate the complexity of food webs. DNA amplification and sequencing techniques of gut and fecal contents are promising approaches, but their success largely depends on the ability to amplify the taxonomic array of prey consumed and then match prey amplicons with reference sequences. When little a priori information on diet is available or a generalist predator is targeted, versatile primer sets (also referred to as universal or general primers) as opposed to group- or species-specific primer sets are the most powerful to unveil the full range of prey consumed. However, versatile primers are likely to preferentially amplify the predominant, less degraded predator DNA if no manipulation is performed to exclude this confounding DNA template. In this study we compare two approaches that eliminate the confounding predator template: restriction digestion and the use of annealing blocking primers. First, we use a preliminary DNA barcode library provided by the Moorea BIOCODE project to 1) evaluate the cutting frequency of commercially available restriction enzymes and 2) design predator specific annealing blocking primers. We then compare the performance of the two predator removal strategies for the detection of prey templates using two versatile primer sets from the gut contents of two generalist coral reef fish species sampled in Moorea. Our study demonstrates that blocking primers should be preferentially used over restriction digestion for predator DNA removal as they recover greater prey diversity. We also emphasize that a combination of versatile primers may be required to best represent the breadth of a generalist''s diet.     

Aversive reactions of two invertebrate predators to European red–black insects

Prey species gain protection by imitating signals of unpalatable models in defensive mimicry. Mimics have been traditionally classified as Batesian (palatable mimic resembling an unpalatable model) or Müllerian (unpalatable mimic resembling a similarly unpalatable model). However, recent studies suggest that rather than discrete categories, the phenomenon of mimicry can be better understood as a continuum. The level of unpalatability of defended prey is a key factor in determining the type of mimetic relationship. Herein, we used insects (ladybugs and true bugs) from a putative European “red–black” mimetic complex as experimental models of defended species and crickets as a control prey. We offered the prey to two species of sympatric invertebrate predators (praying mantis and spider) and video recorded the interactions. We tested three alternative hypotheses, namely (i) the three red–black species tested are similarly defended against both predators; (ii) some red–black species are better defended than others against both predator species, and (iii) the effectiveness of the red–black species defenses is predator dependent. Both predators attacked all prey types with a similar frequency. But while all three red–black species similarly elicited aversive behaviors in spiders, the mantises' aversive reactions varied depending on the prey species. Our results provide support to the third hypothesis, suggesting that the same prey species can fall into different parts of the spectrum of palatability–unpalatability depending on the type of predator.     

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.     

Knowing your enemies: Integrating molecular and ecological methods to assess the impact of arthropod predators on crop pests

The importance of natural enemies as the foundation of integrated pest management (IPM) is widely accepted, but few studies conduct the manipulative field experiments necessary to directly quantify their impact on pest populations in this context. This is particularly true for predators. Studying arthropod predator–prey interactions is inherently difficult: prey items are often completely consumed, individual predator–prey interactions are ephemeral (rendering their detection difficult) and the typically fluid or soft‐bodied meals cannot be easily identified visually within predator guts. Serological techniques have long been used in arthropod predator gut‐contents analysis, and current enzyme linked immunosorbent assays (ELISA) are highly specific and sensitive. Recently, polymerase chain reaction (PCR) methods for gut‐contents analysis have developed rapidly and they now dominate the diagnostic methods used for gut‐contents analysis in field‐based research. This work has identified trophic linkages within food webs, determined predator diet breadth and preference, demonstrated the importance of cannibalism and intraguild predation within and between certain taxa, and confirmed the benefits (predator persistence) and potential disadvantages (reduced feeding on pest species) of the availability of alternative nonpest prey. Despite considerable efforts to calibrate gut‐contents assays, these methods remain qualitative. Available techniques for predator gut‐contents analysis can provide rapid, accurate, cost‐effective identification of predation events. As such, they perfectly compliment the ecological methods developed to directly assess predator impacts on prey populations but which are imperfect at identifying the key predators. These diagnostic methods for gut‐contents analysis are underexploited in agricultural research and they are almost never applied in unison with the critical field experiments to measure predator impact. This paper stresses the need for a combined approach and suggests a framework that would make this possible, so that appropriate natural enemies can be targeted in conservation biological control.