Gut content examination of the citrus predator assemblage for the presence of Homalodisca vitripennis remains

A two-year study was conducted in a citrus orchard, Citrus sinensis L., to determine frequency of predation on glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar). A total of 1,578 arthropod predators, representing 18 taxa, were collected and assayed for the presence of GWSS egg protein by an enzyme-linked immunosorbent assay using a Homalodisca-species and egg-specific monoclonal antibody and then by polymerase chain reaction using a H. vitripennis-specific DNA marker. The gut content analyses revealed the presence of GWSS remains in the gut of 2.28 % of the total arthropod predator population, with 3.09 % of the spiders and 0.59 % of the insect predators testing positive. Moreover, a comparison of the two assays indicated that they were not equally effective at detecting GWSS remains in predator guts. Low frequencies of GWSS detection in the gut of predators indicated that GWSS are not a primary prey and that predators may contribute little to suppression of this pest in citrus.     

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.     

Rapid, high-throughput detection of azalea lace bug (Hemiptera: Tingidae) predation by Chrysoperla rufilabris (Neuroptera: Chrysopidae), using fluorescent-polymerase chain reaction primers

Azalea lace bugs, Stephanitis pyrioides (Scott) (Hemiptera: Tingidae), are the most common pest of azaleas (Rhododendron spp.) in nursery production and the landscape. Although pesticides are commonly used to control lace bugs, natural enemies can be a significant source of lace bug mortality. Lacewings (Neuroptera: Chrysopidae) are natural enemies of lace bugs and easily consume them in laboratory studies. Field studies on lacewing biocontrol of azalea lace bugs are underway; however, monitoring lacewing predation in a nursery environment by direct observation is impractical. Here, we describe a fluorescent-polymerase chain reaction method to estimate S. pyrioides consumption based on the gut contents of lacewing predators. Lace bug DNA was detected in fed lacewings up to 32 h after ingestion. More than 80% of the ingested lace bugs were detected using our method with only one false positive result. The assay is both high-throughput and relatively inexpensive, making it a practical approach to documenting lace bug predation in the field.     

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.     

Identifying inter‐ and intra‐guild feeding activity of an arthropod predator assemblage

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Consumption of cereal leaf beetle,Oulema melanopus,by generalist predators in wheat fields detected by molecular analysis

The cereal leaf beetle (CLB), Oulema melanopus L. (Coleoptera: Chrysomelidae), is a major pest of cereal crops that has recently been reported in western Canada. We developed a set of primers to detect CLB DNA in the gut of six common predator taxa in wheat fields: lady beetles (20 positives of 143 individuals), nabid bugs (73 positives of 206 individuals), and wolf spiders (2 positives of 25 individuals). Nabis americoferus Carayon (Hemiptera: Nabidae) and Coccinella septempunctata L. (Coleoptera: Coccinellidae) were the most abundant predators in cereal fields, with 0.35 and 0.05 proportion of samples positive for CLB DNA, respectively. The prey DNA half-lives were used to adjust the estimates for N. americoferus to 0.22, due to its longer DNA detectability relative to C. septempunctata. Overall, Hippodamia parenthesis (Say) (Coleoptera: Coccinellidae) had the highest proportion of positives at 0.43. There was a positive association between CLB abundance and proportion of N. americoferus and C. septempunctata positives for CLB DNA. This study highlights the contribution of generalist predators to CLB mortality and their important role in integrated management for CLB. Furthermore, we provide a molecular tool that can be used to identify predators of CLB and predation frequency in agricultural fields .     

Delivery of a Genetically Marked <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. to the Glassy-Winged Sharpshooter for Use in a Paratransgenic Control Strategy

An artificial feeding system was designed for the glassy-winged sharpshooter (GWSS), Homalodisca coagulata Say (Hemiptera: Cicadellidae). The system, unlike previous systems, provided enough nutrients to GWSS to survive for 48 h. A system like this is a prerequisite to examining the potential use of paratransgenesis to interrupt transmission of Xylella fastidiosa, the bacterial pathogen causing Pierces disease of grape, by insect vectors. We developed a system for short-term feeding of GWSS that allows for the introduction of bacteria in liquid medium, and we have demonstrated the ability of Alcaligenes xylosoxidans denitrificans, expressing a red fluorescent protein (dsRed), to colonize the cibarial region of the GWSS foregut for up to 5 weeks post-exposure. Alcaligenes xylosoxidans denitrificans thus occupies the same region in the foregut as the pathogen, Xylella fastidiosa.     

Laboratory and field tests of predation on the cereal leaf beetle,Oulema melanopus (Coleoptera: Chrysomelidae)

The cereal leaf beetle (CLB), Oulema melanopus (L.) (Coleoptera: Chrysomelidae), is an invasive pest in North America recently reported in the Canadian Prairies. We performed a series of laboratory assays to identify potential predators and a field study to quantify predation of CLB eggs. In no-choice Petri dish assays, ground beetles (Carabidae), rove beetles (Staphylinidae), and several common lady beetle species (Coccinellidae) were the most consistent predators of eggs and larvae. Nabis spp. (Hemiptera: Nabidae) and wolf spiders (Araneae: Lycosidae) consumed many larvae, but did not consume eggs. Hippodamia spp., Coccinella septempunctata (L.) (Coleoptera: Coccinellidae), and Pterostichus melanarius (Illiger) (Coleoptera: Carabidae) also fed on CLB eggs on potted plants when an alternative food source was available, Sitobion avenae (Fabricius) (Hemiptera: Aphididae). In our field study, we found an average of 24.5% of sentinel eggs disappeared over a 24?h period, likely due to predation. Our results suggest that generalist predators can play an important role in the biological control of CLB, and warrant further study.     

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.     

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.     

First observation of Lethocerus cordofanus (Hemiptera: Belostomatidae) preying on Afronycteris nana (Chiroptera: Vespertilionidae)

Giant water bugs (Hemiptera: Belostomatidae) are key predators in freshwater ecosystems and have been reported to feed on several species of vertebrates, including fishes, amphibians and reptiles. Here, we report the opportunistic predation of an adult female vesper bat (Afronycteris nana) by a giant water bug (Lethocerus cordofanus) in a temporary pond in a rice paddy in Guinea-Bissau, West Africa. To our best knowledge, this is the first instance of natural predation upon a mammal by a giant water bug to be documented in a scientific report.     

Development and preliminary application of a triplex real‐time polymerase chain reaction assay for evaluating predation on three planthoppers in a rice ecosystem

A multiplex real‐time quantitative polymerase chain reaction (PCR) assay was developed to simultaneously detect the DNA of three rice planthoppers, that is, Sogatella furcifera (Horváth) (white‐backed planthopper), Nilaparvata lugens (Stål) (brown planthopper) and Laodelphax striatellus (Fallén) (small brown planthopper), in the gut of their predators. The sets of primers and ALLGlo probes were targeted to the regions of internal transcribed spacer 2 (ITS2) genes in nuclear ribosomal DNA (rDNA). The sensitivity, specificity and interference test for the multiplex real‐time quantitative PCR assay were analysed. The assay's detection limits were 100, 1000 and 100 copies for the white‐backed planthopper, the brown planthopper and the small brown planthopper, respectively. The specificity tests showed no cross‐reactivity with genomic DNA from 30 other dominant herbivores, saprophagous insects and predators from rice ecosystem for each planthopper species. The assay was used in a preliminary study of predation events on the three planthoppers by three major spiders viz., Pardosa pseudoannulata (Bösenberg et Strand), Ummeliata insecticeps (Bösenberg et Strand) and Tetragnatha maxillosa Thorell which each differ in their preferred microhabitat as well as their predatory habits in rice field, and the results showed their predation on each planthopper species could be well evaluated using this method. Therefore, the multiplex real‐time quantitative PCR assay provides a new tool to study the mechanisms of prey shifting and natural regulation of the three rice planthoppers by generalist predators in rice ecosystem.     

Sticky predators: a comparative study of sticky glands in harpactorine assassin bugs (Insecta: Hemiptera: Reduviidae)

Zhang, G. and Weirauch, C. 2011. Sticky predators: a comparative study of sticky glands in harpactorine assassin bugs (Insecta: Hemiptera: Reduviidae). —Acta Zoologica (Stockholm) 00 : 1–10. For more than 50 years, specialized dermal glands that secrete sticky substances and specialized setae have been known from the legs of New World assassin bugs in the genus Zelus Fabricius (Reduviidae: Harpactorinae). The gland secretions and specialized ‘sundew setae’ are involved in enhancing predation success. We here refer to this predation strategy as ‘sticky trap predation’ and the specialized dermal glands as ‘sticky glands’. To determine how widespread sticky trap predation is among Reduviidae, we investigated taxonomic distribution of sticky glands and sundew setae using compound light microscopical and scanning electron microscopical techniques and sampling 67 species of Reduviidae that represent 50 genera of Harpactorini. We found sticky glands in 12 genera of Harpactorini and thus show that sticky trap predation is much more widespread than previously suspected. The sticky glands vary in shape, size and density, but are always located in a dorsolateral position on the fore tibia. Sundew setae are present in all taxa with sticky glands with the exception of Heza that instead possesses unique lamellate setae. The sticky trap predation taxa are restricted to the New World, suggesting a New World origin of this unique predation strategy.     

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.     

The influence of time and temperature on molecular gut content analysis： Adalia bipunctata fed with Rhopalosiphum padi

Gut content analysis is a useful tool when studying arthropod predator-prey interactions. We used polymerase chain reaction （PCR） technique to examine how detection of prey DNA in the gut content of predators was influenced by digestion time and temperature. Such knowledge is critical before applying PCR-based gut content analysis to field collected predators. Larvae of the two-spotted ladybeetle （Adalia bipunctata L.） were fed with the bird cherry-oat aphid （Rhopalosiphum padi L.） at either 21℃ or 14℃. After consuming one aphid, the predators were allowed to digest the prey for a range of time periods up to 24 hours. The influence of temperature on A. bipunctata feeding behavior was also recorded. From the fed larvae, total DNA was extracted and PCR reactions with R. padi specific primers were run. The number ofA. bipunctata that tested positive for R. padi DNA was negatively related to the length of digestion time. Temperature influenced larval feeding behavior but did not have a significant effect on R. padi DNA detection. After pooling the data from both temperature treatments we estimated the time point when R. padi DNA could be amplified from 50% of the fed A. bipunctata by PCR to be 4.87 hours. With such a rapid decrease in prey DNA detection success, positive PCR reactions will most likely be the result of predation events occurring shortly before capture. If a defined digestion temperature range has proven not to influence prey detection, PCR data obtained from predators collected within that particular range can be interpreted in the same way.     

Determining the frequency of heteropteran predation on sweetpotato whitefly and pink bollworm using multiple ELISAs

The gut contents of field-collected, predaceous Heteroptera were assayed for the presence of eggs of the sweetpotato whitefly,Bemisia tabaci Gennadius (Homoptera: Aleyrodidae) and the pink bollworm,Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) using multiple enzyme-linked immunosorbent assays (ELISAs). Of seven species examined,Geocoris species andOrius tristicolor (Say) were the most frequent predators of sweetpotato whitefly with 32–39% of the individuals tested over the whole season scoring positive for whitefly antigens. With the exception ofLygus hesperus Knight, a major insect pest as well as a predator, the frequency of predation on pink bollworm eggs was much lower (0.7–14.3% positive over the season). Relatively few predators tested positive for both antigens (0.3–12.5%).     

Ten real‐time PCR assays for detection of fish predation at the community level in the San Francisco Estuary–Delta

The effect of predation on native fish by introduced species in the San Francisco Estuary–Delta (SFE) has not been thoroughly studied despite its potential to impact species abundances. Species‐specific quantitative PCR (qPCR) is an accurate method for identifying species from exogenous DNA samples. Quantitative PCR assays can be used for detecting prey in gut contents or faeces, discriminating between cryptic species, or detecting rare aquatic species. We designed ten TaqMan qPCR assays for fish species from the SFE watershed most likely to be affected by non‐native piscivores. The assays designed are highly specific, producing no signal from co‐occurring or related species, and sensitive, with a limit of detection between 3.2 and 0.013 pg/μL of target DNA. These assays will be used in conjunction with a high‐throughput qPCR platform to compare predation rates between native and non‐native piscivores and assess the impacts of predation in the system.     

Spider predation on a mirid pest in Japanese rice fields

Spiders are common generalist predators, and understanding their potential in biological control is important for the development of integrated pest management programs. In this study, predation by three groups of spiders on the mirid bug Stenotus rubrovittatus (Hemiptera: Miridae) in rice paddies was investigated using DNA-based gut-content analysis. A laboratory feeding study revealed that the detection half-lives of bug DNA in the spider gut at 25 °C was 3.4 days for Lycosidae and 1.5 days for Tetragnathidae. Individual spider predation on the mirid bug was investigated by detecting DNA of prey in field-collected spiders. In total, 1199 spiders were assayed from three spider groups: Pirata subpiraticus (Lycosidae), Tetragnatha spp. (Tetra-gnathidae), and Pachygnatha clercki (Tetra-gnathidae), which each differ in their preferred microhabitat as well as their predatory habits. Detection rates of prey DNA in spiders increased significantly with the density of prey across all spider groups. P. subpiraticus and Tetragnatha spp. predation showed a better fit to a saturated response curve to increasing prey density, while P. clercki showed a simple linear relationship with prey density. Densities of alternative prey species did not affect the detection rates of mirids. These results suggest that predation on pests by generalist predators in an agroecosystem is affected not only by prey abundance but also by predator preference for specific prey. Predator preference is therefore an important factor to consider when estimating the role of natural enemies as biological control agents.     

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.     

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.