An immunological approach to quantify consumption of protein-tagged Lygus hesperus by the entire cotton predator assemblage

A new method for post-mortem quantification of predation on prey items marked with protein antigens is described. First, short-term protein marking retention tests were conducted on the targeted prey, immature Lygus hesperus Knight (Heteroptera: Miridae). Chicken IgG, rabbit IgG, or soy milk proteins were readily detectable by a suite of protein specific enzyme-linked immunosorbent assays (ELISA) on the L. hesperus. Then, predator gut content assays were conducted on chewing and piercing–sucking type predators that consumed a 3rd instar L. hesperus marked with rabbit IgG. The rabbit IgG gut content ELISA detected the marked prey in the vast majority of both types of predators for up to 24 h after feeding. Finally, field cage studies were conducted to quantify predation rates of the natural cotton predator assemblage on protein marked L. hesperus nymphs. Each 4th instar L. hesperus marked with rabbit IgG, chicken IgG, and soy milk was released into one of 360 field cages containing a cotton plant and the natural population of predators. After 7 h, each caged plant was pulled from the field, the number of predaceous arthropods in each cage were tallied, and each individual predator was assayed for the presence of marked prey by a suite of protein-specific ELISAs. A procedural error with the soy mark application negated the anti-soy ELISA data, but the anti-rabbit IgG and anti-chicken IgG ELISAs pinpointed exactly which predators preyed on the IgG marked nymphs. The protein-specific gut ELISAs revealed that various members of Araneae, Heteroptera, and Coleoptera were the most common predators of the marked prey items. In all, 74 predation events were recorded in the guts of the 556 predators encountered in the field cages. Of these 26, 23, and 14 marked individuals were eaten by various members of Araneae, Heteroptera, and Coleoptera, respectively. This study verifies that prey immunomarking is a simple, versatile, and effective method for quantifying predation rates on L. hesperus.     

Standardization of prey immunomarking: does a positive test always indicate predation?

A prey immunomarking procedure (PIP) in combination with generic anti-rabbit and anti-chicken immunoglobulin G (IgG) enzyme-linked immunosorbent assays (ELISAs) are used frequently to study arthropod predation. This study was conducted to: (1) further standardize the PIP as a tool for predator gut analysis research, (2) investigate the most effective means for administering IgG marks to prey items, and (3) assess the possibility of the PIP yielding false positive reactions as a consequence of a predator obtaining a mark by incidental contact with, or by a failed predation attempt on, a protein-marked prey item. The pest Lygus hesperus Knight (Hemiptera: Miridae) was tagged with either an external rabbit IgG mark, an internal chicken IgG mark, or a double (external rabbit IgG and internal chicken IgG) mark treatment. Then, the variously marked prey items were fed to chewing and piercing-sucking type predators and their gut contents were examined for the presence of IgG remains. Data revealed that all three marking treatments were highly effective at tagging targeted prey. However, ELISA results showed that the prey items should only be marked internally to maximize the likelihood of detecting prey remains while minimizing the risk of obtaining false positive errors. The merits and limitations of using the generic PIP for predator gut analysis research are discussed.     

Development of an immunological technique for identifying multiple predator–prey interactions in a complex arthropod assemblage

A simplified but highly effective approach for the post‐mortem evaluation of predation on several targeted members of an arthropod assemblage that does not require the development of pest‐specific enzyme‐linked immunosorbent assay (ELISA) (e.g. pest‐specific monoclonal antibodies) or PCR assays (DNA primers) is described. Laboratory feeding studies were conducted to determine if predation events could be detected from predators that consumed prey marked with foreign protein. I determined that large and small rabbit immunoglobulin G (IgG)‐marked prey can be detected by a rabbit‐IgG‐specific ELISA in the guts of chewing and piercing–sucking type predators. I then conducted multifaceted inclusion and exclusion field cage studies to qualify the degree of interguild and intraguild predation occurring among a complex arthropod assemblage during four separate light phase treatments. The field cages contained an arthropod assemblage consisting of 11 or 12 species of predaceous arthropods and three pest species. The three pests introduced into the cages included third instar Trichoplusia ni marked with rabbit IgG, third instar Lygus hesperus marked with chicken IgG and Pectinophora gossypiella sentinel egg masses. The inclusion cages allowed foraging fire ants, Solenopis xyloni, to freely enter the cages while the exclusion cages contained barriers that prevented ant entry. The results obtained using the conventional inclusion/exclusion field cage methodology revealed that there was substantial interguild and intraguild predation occurring on the majority of the arthropods in the assemblage, particularly in those cages that included ants. I then precisely identified which predators in the assemblage were feeding on the three targeted pests by conducting three post‐mortem gut content analyses on each individual predator (1503 individuals) in the assemblage. Specifically, P. gossypiella egg predation events were detected using an established P. gossypiella‐egg‐specific ELISA, and third instar T. ni and L. hesperus predation events were detected using rabbit‐IgG‐specific and chicken‐IgG‐specific ELISAs, respectively. Generally, the gut ELISAs revealed that Collops vittatus, Spanagonicus albofasciatus and Geocoris punctipes readily preyed on P. gossypiella eggs; Nabis alternatus, Zelus renardii and spiders (primarily Misumenops celer) readily preyed on marked L. hesperus nymphs, and spiders, S. albofasciatus and N. alternatus readily preyed on T. ni larvae. Furthermore, the cage methods and the post‐mortem predator gut ELISAs revealed very few distinctive patterns of predation with regard to the light cycle the assemblage was exposed to.     

Factors affecting detectability of prey DNA in the gut contents of invertebrate predators: a polymerase chain reaction-based method

This study aimed to determine factors that influence the detection of DNA of Plutella xylostella L. (Lepidoptera: Plutellidae) in the gut contents of arthropod predators when the polymerase chain reaction is used to amplify a diagnostic fragment of the gene coding for cytochrome oxidase subunit I. The effects of temperature, time since feeding, subsequent food intake, sex, weight, and species of predator on prey detectability were studied in the laboratory. Three types of predator were studied: the spider Venator spenceri Hogg. (Araneae: Lycosidae), a bug with sucking mouthparts, Nabis kinbergii (Reuter) (Heteroptera: Nabidae), and a coccinellid with chewing mouthparts, Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae). In all experiments, the detectability of prey DNA was negatively correlated with time post‐feeding. The duration of detectability differed among the predator species. The time calculated for median detection success at 20 °C ranged from 49.6 h in V. spenceri to 36.1 h in N. kinbergii and 17.1 h in H. variegata. In H. variegata, but not in V. spenceri, the rate of detection decreased with increasing temperature. Subsequent food intake did not affect the detectability of DNA of P. xylostella in V. spenceri. In H. variegata, sex and weight of the predator did not influence detection of prey DNA. In addition, this study uncovered potential sources of error caused by detection of prey DNA following secondary cannibalistic and intraguild predation. The results provide essential information for the interpretation of prey detection data from field‐collected predators’ gut contents.     

An immunological approach to distinguish arthropod viviphagy from necrophagy

Scavenging activity of predators inhabiting agroecosystems has not been thoroughly investigated. Understanding the prevalence of necrophagy in predators is paramount to determining the effectiveness of biological control agents. A molecular predator gut content assay is described that can differentiate necrophagy from viviphagy. Cadaver sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) and green lacewing, Chrysoperla rufilabris Burmeister (Neuroptera: Chrysopidae) serving as targeted prey items were marked with rabbit immunoglobulin G (IgG) protein and live prey items were marked with chicken IgG, respectively. The marked prey items were fed to convergent lady beetles, Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) and soft-winged flower beetles, Collops vittatus (Say) (Coleoptera: Melyridae). The frequency of detection of the protein-marked prey items in the gut of the predaceous beetles was assessed at 0, 3, 6, 12, 24 and 48 h after feeding using a rabbit-IgG-specific or chicken-IgG-specific enzyme-linked immunosorbent assay (ELISA). Each IgG-specific ELISA detected the presence of the marker proteins in the gut of 90 % of the predators up to 12 h after prey consumption. A laboratory feeding study was also conducted to determine the propensity that each predator species engages in viviphagy and necrophagy. The laboratory feeding observations revealed that C. vittatus prefer carrion prey items. Finally, the laboratory observations of necrophagy were confirmed in a field study where C. vittatus was observed, directly and indirectly, feeding on H. convergens carcasses. The methodologies described here are useful for future studies on various aspects of insect predation.     

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.     

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).

     

A comparative study on the efficacy of a pest-specific and prey-marking enzyme-linked immunosorbent assay for detection of predation

The efficacy of two different antigen–antibody combinations to detect predation on eggs of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) was compared. The first method was an indirect enzyme‐linked immunosorbent assay (ELISA) using monoclonal antibody‐based gut content analysis that detects H. armigera egg protein. The second method was a sandwich ELISA that detects an exotic protein [rabbit immunoglobulin G (IgG)] applied as an external marker to H. armigera eggs. The target predators were the predatory beetles Dicranolaius bellulus (Guerin‐Meneville) (Coleoptera: Melyridae) and Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae). Beetles were fed with H. armigera eggs that had been marked with rabbit IgG and then held at various intervals after prey consumption. Each individual beetle was then assayed by both ELISA techniques to identify the prey remains in their guts. The two ELISA methods were further tested on field‐collected predators. Specifically, protein‐marked egg masses were strategically placed in a cotton field. Then, predators from surrounding cotton plants were collected at various time intervals after the marked eggs were exposed and assayed by both ELISAs to detect the frequency of predation on the marked eggs. The rabbit IgG‐specific sandwich ELISA had a higher detection rate than the H. armigera‐specific indirect ELISA under controlled and field conditions for both predator species. Moreover, a greater proportion of field‐collected D. bellulus tested positive for predation than H. variegata. The advantages and disadvantages of using prey‐marking ELISAs instead of pest‐specific ELISA assays are discussed.     

Does dimethyl sulfoxide increase protein immunomarking efficiency for dispersal and predation studies?

Marking biological control agents facilitates studies of dispersal and predation. This study examines the effect of a biological solvent, dimethyl sulfoxide (DMSO), on retention of immunoglobulin G (IgG) protein solutions applied to Diorhabda carinulata (Desbrochers) (Coleoptera: Chrysomelidae), an important biological control agent of saltcedar, either internally by feeding them protein‐labeled foliage or externally by immersing them in a protein solution. In addition, we determined whether internally or externally marked DMSO‐IgG labels could be transferred via feeding from marked D. carinulata to its predator, Perillus bioculatus (Fabricius) (Heteroptera: Pentatomidae). The presence of rabbit and chicken IgG proteins was detected by IgG‐specific enzyme‐linked immunosorbent assays (ELISA). DMSO‐IgG treatments showed greater label retention than IgG treatments alone, and this effect was stronger for rabbit IgG than for chicken IgG. Fourteen days after marking, beetles immersed in rabbit IgG showed 100% internal retention of label, whereas beetles immersed in chicken IgG showed 65% internal retention. Immersion led to greater initial (time 0) label values, and longer label retention, than feeding beetles labeled foliage. The DMSO‐IgG label was readily transferred to P. bioculatus after feeding on a single marked prey insect. This investigation shows that addition of DMSO enhances retention of IgG labels, and demonstrates that protein marking technology has potential for use in dispersal and predator–prey studies with D. carinulata. Moreover, our observation of P. bioculatus feeding on D. carinulata is, to our knowledge, a new predator–prey association for the stink bug.     

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.     

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.     

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.     

Use of a gut content ELISA to detect whitefly predator feeding activity after field exposure to different insecticide treatments

A 2-year commercial-scale study was conducted to qualitatively evaluate the effect of different insecticide treatment regimes on the predator complex attacking Bemisia tabaci (Gennadius) in cotton. In 1996 three insecticide regimes were compared: a rotation of conventional broad-spectrum insecticides or one of two different regimes based on the initial use of the insect growth regulators (IGRs), buprofezin and pyriproxyfen. In 1997 the same three regimes plus an untreated control were compared; split-plots were sprayed once for Lygus hesperus Knight control using a broad-spectrum insecticide. Relative feeding activity for each predator species was compared between treatment regimes by analyzing the gut contents of predators for the presence of whitefly remains using a whitefly-specific enzyme-linked immunosorbent assay (ELISA). The ELISA results were combined with predator density data to obtain a qualitative pesticide impact index for each predator group. In total, we analyzed the gut contents of 32 262 field-collected predators, representing nine different taxa. Of these, Pseudatomocelis seriatus (Reuter), Spanagonicus albofasciatus (Reuter), and spiders consisting primarily of Misumenops celer (Hentz) are shown to be whitefly predators for the first time. Predator populations were usually reduced in plots that received applications of broad-spectrum insecticides for B. tabaci and L. hesperus control, but there were few treatment differences in the proportions of predators containing whitefly remains in their guts. However, the feeding activity of certain predator species in fields sprayed with broad spectrum insecticides was significantly reduced compared with those in IGR-based and control treatments. Overall, insecticide regimes using IGRs were less lethal to the whitefly predator complex than regimes consisting of only conventional, broad-spectrum insecticides, but differences in predator feeding activity on whitefly between the various insecticide treatment regimes were minimal.     

Gut microbiomes of mobile predators vary with landscape context and species identity

Landscape context affects predator–prey interactions and predator diet composition, yet little is known about landscape effects on insect gut microbiomes, a determinant of physiology and condition. Here, we combine laboratory and field experiments to examine the effects of landscape context on the gut bacterial community and body condition of predatory insects. Under laboratory conditions, we found that prey diversity increased bacterial richness in insect guts. In the field, we studied the performance and gut microbiota of six predatory insect species along a landscape complexity gradient in two local habitat types (soybean fields vs. prairie). Insects from soy fields had richer gut bacteria and lower fat content than those from prairies, suggesting better feeding conditions in prairies. Species origin mediated landscape context effects, suggesting differences in foraging of exotic and native predators on a landscape scale. Overall, our study highlights complex interactions among gut microbiota, predator identity, and landscape context.     

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.     

Partial consumption of prey: the significance of prey water loss on estimates of biomass intake

Summary A number of studies on the feeding behaviour of sucking predators have estimated the weight of biomass the predator extracts from the prey by measuring the weight change occurring in the prey. This method does not consider that a proportion of the prey weight change is lost to the immediate environment. I examined the spider Diaea sp. feeding on the fruit fly Drosophila immigrans and found that the prey lost approximately 28% more weight than the predator gained. This difference was largely explained by water loss from the prey. My results suggest that water loss, which is not available to the predator, is an important part of prey weight loss. To avoid overestimating predator biomass gain it is necessary to measure the predator weight gain directly or take into account water loss as a component of prey weight change.     

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.     

A false‐positive food chain error associated with a generic predator gut content ELISA

Conventional prey‐specific gut content ELISA (enzyme‐linked immunosorbent assay) and PCR (polymerase chain reaction) assays are useful for identifying predators of insect pests in nature. However, these assays are prone to yielding certain types of food chain errors. For instance, it is possible that prey remains can pass through the food chain as the result of a secondary predator (hyperpredator) consuming a primary predator that had previously consumed the pest. If so, the pest‐specific assay will falsely identify the secondary predator as the organism providing the biological control services to the ecosystem. Recently, a generic gut content ELISA was designed to detect protein‐marked prey remains. That assay proved to be less costly, more versatile, and more reliable at detecting primary predation events than a prey‐specific PCR assay. This study examines the chances of obtaining a ‘false positive’ food chain error with the generic ELISA. Data revealed that the ELISA was 100% accurate at detecting protein‐marked Lygus hesperus Knight (Hemiptera: Miridae) remains in the guts of two (true) primary predators, Hippodamia convergens Guérin‐Méneville (Coleoptera: Coccinellidae) and Collops vittatus (Say) (Coleoptera: Melyridae). However, there was also a high frequency (70%) false positives associated with hyperpredators, Zelus renardii Kolenati (Hemiptera: Reduviidae), that consumed a primary predator that possessed protein‐marked L. hesperus in its gut. These findings serve to alert researchers that the generic ELISA, like the PCR assay, is susceptible to food chain errors.     

Are sweep net sampling and pitfall trapping compatible with molecular analysis of predation?

Molecular analysis of predation enables accurate and reliable elucidation of trophic linkages in complex food webs, but identifying the strength of such interactions can be subject to error. Currently two techniques dominate: monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Although the optimization and characterization of these systems ensures their sensitivity and specificity, predator collection protocols such as sweep-netting and vacuum sampling could overestimate feeding rates because of surface-level contamination, yielding positive reactivity or predation within the sampling device. Therefore, two sampling techniques (sweep-net sampling and hand collection) were compared within an alfalfa agroecosystem using a monoclonal antibody-based ELISA to test the hypothesis that cross-contamination is a source of error, i.e., significantly more predators (linyphiid spiders) would test positive for prey (Diptera) proteins. A concurrent study examining the viability of trapping predators into saline solution was also undertaken. No significant differences were found between the proportions of spiders screening positive for Diptera when collected by sweep-net versus hand collection, rejecting the hypothesis that sweep-netting predators for subsequent molecular gut content analysis overestimates predation frequency. ELISA was also capable of detecting prey proteins in predator guts from pitfall traps containing phosphate-buffered saline, indicating the suitability of this approach for the collection and analysis of epigeal predators. Although these results indicate that sweep netting and pitfall trapping into solution is appropriate in this predator-prey and ELISA analysis system, caution should be exercised with other interactions and PCR-based analysis. The likelihood for false-positive reactivity should therefore be considered on a case-by-case basis.     

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.