DNA-based Gut Content Analysis Research Articles

Trap crops enhance the control efficacy of Metarhizium brunneum against a soil-dwelling pest

Soil-dwelling insect pests may cause considerable damage to crops worldwide, and their belowground lifestyle makes them hard to control. Amongst the most promising control agents for subterranean pests are soilborne entomopathogenic fungi (EPF) such as Metarhizium brunneum. Albeit EPF can be highly pathogenic to their target pest species under laboratory conditions, their efficacy in the field is often limited due to adverse environmental conditions. Here, we test for the first time if the efficacy of EPF can be improved when they are augmented with trap crops. In a field experiment, the M. brunneum strain ART2825 was combined with a trap crops mixture of six plant species and evaluated for its control effect of wireworms (Coleoptera: Elateridae). When both were combined in the main crop, potato damage was lowered on average by 42.5% and wireworm abundance by 50.8%. Single application of trap crops or EPF lowered damage/pest abundance only by 29.9%/15.89% and 34.7%/4.77%, respectively. Importantly, the strength of the synergistic pest control effect between trap crops and EPF increased disproportionately with increasing wireworm abundance. However, DNA-based gut content analysis showed that wireworms’ feeding preferences were not shifting toward the trap crops. Our findings demonstrate that combining trap crops with EPF improves the efficacy of the latter and leads to a synergistic control effect which magnifies with increasing wireworm abundance. Hence, the synergistic effect of EPF and trap crops might be a promising control strategy for soil-dwelling pests in general and significantly improve our abilities to manage soil pests environmentally friendly.

Identifying plant DNA in the sponging–feeding insect pest Drosophila suzukii

Drosophila suzukii (Diptera: Drosophilidae) is a highly polyphagous invasive pest threatening fruit production in the Americas and Europe. The current knowledge of its host plants is mainly based on oviposition and larval development in fruits, while little is known on the diet of the adult flies. This information is important for developing effective control strategies. Here, we examine DNA-based techniques to determine food plants of D. suzukii. Adult flies were fed with raspberries (Rubus idaeus) and allowed to digest up to 72 h after feeding. Raspberry DNA was detected by diagnostic PCR for up to 48 h post-feeding with a significant negative effect of time on DNA detection success but no significant differences between male and female flies in detection probabilities. As D. suzukii walks on plants, its body surface can get contaminated with DNA. With a bleaching experiment, we succeeded to remove contaminating external plant DNA, while the DNA in the gut content stayed unaffected. Finally, field-collected flies were subjected to a next-generation sequencing approach, demonstrating that plant DNA from different host plants can be efficiently detected in both bleached and non-bleached specimens. In order to safeguard against erroneous host plant detections, we recommend bleaching flies before they are subjected to DNA extraction. The current findings encourage the use of DNA-based gut content analysis in D. suzukii to obtain a better understanding of its feeding ecology which is a prerequisite for developing successful control strategies.

Molecular detection of field predation among larvae of two ladybird beetles is partially predicted from laboratory experiments

Despite the fact that natural enemies can synergistically contribute to herbivore pest suppression, sometimes predators engage in intraguild predation (IGP) that might dampen trophic cascades. DNA-based gut-content analysis has become common in assessing trophic connections and biocontrol potential by predators in field systems. Here, we developed a molecular technique that can be used to unravel predation among two ladybirds, Coccinella septempunctata and Hippodamia variegata, and their shared prey, Aphis gossypii. Both ladybirds may provide effective control of the pest. Therefore, understanding their likelihood to engage in IGP is crucial for conservation biological control. Ladybird specimens were collected in melon crop. DNA extraction, primer design and evaluation were conducted. Detectability of prey DNA did not differ significantly between the two ladybirds. H. variegata exhibited higher predation on A. gossypii than C. septempunctata (90.6% vs. 70.9%) and data correction based on DNA detectability confirmed this ranking. IGP was similar among the two species, although corrected data might suggest a stronger predation by C. septempunctata. Intriguingly, IGP by C. septempunctata was lower than predicted by laboratory bioassays, possibly due to the high complexity that arises under field conditions. Implications of our results for biological control and perspectives for ecological network analysis are discussed.

Overcoming PCR Inhibition During DNA-Based Gut Content Analysis of Ants.

Generalist predators play an important role in many terrestrial systems, especially within agricultural settings, and ants (Hymenoptera: Formicidae) often constitute important linkages of these food webs, as they are abundant and influential in these ecosystems. Molecular gut content analysis provides a means of delineating food web linkages of ants based on the presence of prey DNA within their guts. Although this method can provide insight, its use on ants has been limited, potentially due to inhibition when amplifying gut content DNA. We designed a series of experiments to determine those ant organs responsible for inhibition and identified variation in inhibition among three species (Tetramorium caespitum (L.), Solenopsis invicta Buren, and Camponotus floridanus (Buckley)). No body segment, other than the gaster, caused significant inhibition. Following dissection, we determined that within the gaster, the digestive tract and crop cause significant levels of inhibition. We found significant differences in the frequency of inhibition between the three species tested, with inhibition most evident in T. caespitum The most effective method to prevent inhibition before DNA extraction was to exude crop contents and crop structures onto UV-sterilized tissue. However, if extracted samples exhibit inhibition, addition of bovine serum albumin to PCR reagents will overcome this problem. These methods will circumvent gut content inhibition within selected species of ants, thereby allowing more detailed and reliable studies of ant food webs. As little is known about the prevalence of this inhibition in other species, it is recommended that the protocols in this study are used until otherwise shown to be unnecessary.

Molecular tracking of arthropod predator–prey interactions in Mediterranean lettuce crops

The feeding habits of the generalist arthropod predators in agroecosystems are often difficult to determine, as they are small, mobile and live among the vegetation or in the soil. DNA-based gut-content analysis is a powerful tool that enables the study of arthropod predator–prey interactions. Predation on two of the main pests of Mediterranean lettuce crops, the lettuce aphid, Nasonovia ribisnigri, and the western flower thrips, Frankliniella occidentalis, as well as on Collembola, the most abundant non-pest prey, was studied. Generalist arthropods, like hoverflies, anthocorids (Orius spp.), coccinellids and spiders were collected in lettuce plots in two seasons (spring and summer) and analysed by conventional PCR using N. ribisnigri, F. occidentalis and Collembola-specific primers. Our results showed that in spring the main pest was N. ribisnigri, which was consumed by hoverfly larvae and coccinellids. In summer, the main pest was F. occidentalis, which was mainly predated by Orius spp. followed by hoverfly larvae. Spiders, which fed mainly on Collembola, did not seem to contribute to control of either target pest. This study offers a deeper knowledge of the trophic relationships present in Mediterranean lettuce crops, laying the groundwork for implementing biological control programmes based on the conservation of natural enemies.

Detection of DNA in Aphid Honeydew: A Source of Error for Molecular Gut Content Analysis?

Abstract Aphids (Hemiptera: Aphididae) are pests of a vast array of crops globally, contributing to significant yield loss annually. Biological control, utilizing natural enemies to suppress focal pest species, has often been integrated into aphid management practices. To ensure that arthropod predators are consuming the target aphid, and ultimately have an impact on pest population dynamics, it is important to gather information on consumption patterns in space and time. Molecular gut content analysis can be used to assess the strength of trophic linkages between predators and prey by detecting the presence of species-specific DNA (including aphid DNA) in their gut. However, many generalist predators readily consume aphid honeydew as an additional nutritive resource beyond direct consumption of prey. This raises the important question of whether aphid honeydew is responsible for false-positive predation events being recorded using DNA-based gut content analysis, should honeydew contain detectable quantit...

Mutualistic and antagonistic trophic interactions in canola: The role of aphids in shaping pest and predator populations

Aphids have important effects on the abundance and occurrence of tending ants, predators, and pests in agronomic systems, and DNA-based gut content analysis can aid in establishing predator–prey interactions. The purpose of this study was to determine how the presence of aphids, ants, and pest individuals interact within canola (syn. oilseed rape) fields. Using seasonal data from canola fields, the relationships among ants, aphids and pest individuals were determined, along with the use of PCR techniques in order to amplify aphid DNA and confirm food web links on predators who consume aphids. We determined that aphid presence positively influences the number of ants and predators in a community, and diminishing aphid populations over the growing season were associated with declines in both ants and predators. These reduced populations of predators and aphids may have provided the opportunity for a key pest, Pieris rapae to build populations as the season ensued. This research suggests that complex interactions among herbivores and shared predators contribute to pest outbreaks.

Consumption of flea beetles (Phyllotreta, Coleoptera: Chrysomelidae) by spiders in field habitats detected by molecular analysis

AbstractFlea beetles, Phyllotreta Chevrolat (Coleoptera: Chrysomelidae) species, are often found in oilseed rape (OSR), Brassica napus Linnaeus (Brassicaceae). Among predators in the generalist predator complex present in agricultural fields, wolf spiders (Araneae: Lycosidae) are found on the ground and cobweb spiders (Araneae: Theridiidae) build webs in the foliage. We present group-specific primers developed for five flea beetle species within the genus Phyllotreta and study the incidence of predation of flea beetles by these spider groups using DNA-based gut-content analysis. Wolf spiders of the genus Pardosa Koch and the cobweb spider, Phylloneta impressa (Koch), were collected in three winter OSR fields. Flea beetle densities as well as the occurrence of predators and alternative prey were monitored. In total 19.4% of the collected Pardosa tested positive for flea beetle DNA in the polymerase chain reaction analyses, whereas 10% P. impressa were positive. Pardosa were more likely to be positive for flea beetle DNA when Pardosa activity density was low. Phylloneta impressa were more likely to be positive for flea beetle DNA if they were positive for pollen beetle DNA. Implications of these results for conservation biological control and future studies of food webs in OSR are discussed.

The effect of predator identity on post-feeding prey DNA detection success in soil-dwelling macro-invertebrates

Soils harbour an extraordinary diversity of animals which are functionally connected by their feeding interactions. Determining these feeding links, however, is difficult due to the opaque habitat the animals live in and because many prey remains found in predator gut contents cannot be identified morphologically. DNA-based gut content analysis overcomes these hurdles. This approach opens new possibilities to empirically examine below-ground feeding links by measuring prey DNA detection frequencies in various predators. Comparing prey DNA detection rates between different predator taxa, however, is not straightforward as digestion rates and thus post-feeding prey DNA detection intervals can be affected by predator identity. In the present study we examined the effect of predator identity for a range of soil-dwelling adult and larval beetles as well as lithobiid and geophilid centipedes. These predators were fed with white grubs (scarabaeid beetle larvae) and their post-feeding prey DNA detection intervals were established for small, medium and large prey DNA fragments. In geophilids the 50% detection probability for the medium prey DNA fragment was 98 h post-feeding whereas it ranged between 22 and 34 h in beetles. Among the different beetle species, the magnitude of the differences in detection intervals was strongly affected by the size of the prey DNA fragment targeted: the intervals showed the highest similarity in the medium-sized prey DNA fragment which was most frequently detected. Predator biomass and meal size rarely affected post-feeding prey DNA detection intervals. Our findings suggest that prey DNA detection success is similar within taxonomically related species of soil-dwelling macro-invertebrates such as beetles and centipedes. Between these groups prey detection can be adjusted according to taxon-specific digestion rates for better comparisons. Moreover, the magnitude of the predator identity effect is modulated by assay sensitivity: highly sensitive assays which target small- to medium-sized prey DNA fragments are recommended as these minimize the interspecific differences in post-feeding prey DNA detection intervals.

Feeding habits of lycosid spiders in field habitats

Generalist arthropod predators are potential drivers of population dynamics in a wide variety of ecosystems but their feeding habits are often difficult to reveal as they are small, mobile, and live among dense vegetation or in soils. DNA-based gut-content analysis is a powerful tool that enables studies on arthropod predator–prey interactions. We studied lycosid spiders (Pardosa spp.) in agroecosystems to see if they consumed cereal aphids (Rhopalosiphum padi) and Collembolans at random, i.e., in proportion to their abundance in the field. We also tested if consumption of the target prey items was affected by the presence of alternative food. Spiders were captured in farmers’ fields and their gut-contents screened by PCR with R. padi and Collembola primers. On all sampling occasions, concurrent assessments of total prey availability were carried out. Spider predation rates on R. padi always exceeded 50 %. Spiders also tested positive for Collembola but to a lower and more varying degree. In general, Pardosa did not consume R. padi and Collembolans in relation to their abundance in the field. Aphid predation was much higher than expected whereas consumption of Collembolans was considerably lower. The presence of alternative prey influenced consumption of the aphid. It was concluded that prey consumption by Pardosa spiders generally cannot be assumed to simply mirror prey availability. The spatial distribution of the target prey needs to be considered as well as the abundance, composition, and nutritional content of potential alternative food items.

DNA‐based analysis of regurgitates: a noninvasive approach to examine the diet of invertebrate consumers

DNA-based gut content analysis has become an important tool for unravelling feeding interactions in invertebrate communities under natural conditions. It usually implies killing of the consumer and extracting the DNA from its food, using either the whole animal or its dissected gut. This post-mortem approach, however, is not suitable for investigating the diet of rare or protected species and also prohibits tracking individual dietary preferences as each consumer can provide trophic information only once. Moreover, removing large numbers of consumers from a habitat for analysis might critically change population densities and affect species interactions. Here, we present DNA-based analysis of invertebrate regurgitates, a novel approach to overcome these limitations. Conducting feeding experiments where adult Poecilus cupreus (Coleoptera: Carabidae) were fed with larvae of Amphimallon solstitiale (Coleoptera: Scarabaeidae), we show that detection success in regurgitates compared to samples prepared from whole beetles was similar or significantly enhanced for small/medium and large prey DNA fragments, respectively. Prey DNA detection success remained high in regurgitates stored in ethanol for 21 months at room temperature prior to DNA extraction. We conclude that in those invertebrates where regurgitates can be obtained, examination of food DNA in regurgitates offers many advantages over conventional post-mortem gut content analysis.

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.

Scavenging and active predation in generalist predators: A mesocosm study employing DNA-based gut content analysis

Abstract Scavenging presumably is common among invertebrate generalist predators, however, only few studies have specifically dealt with the consumption of dead prey. Here consumption of dead and living prey within a guild of generalist predators was investigated using mesocosm experiments and DNA-based gut content analysis. A community of predators including carabids, staphylinids and spiders was released into 0.2 m 2 mesocosms, planted with wheat and infested with grain aphids Sitobion avenae . At predator release, freshly killed bird cherry-oat aphids, Rhopalosiphum padi , were provided as carrion prey at the soil surface. Two days after predator release, predators were collected and their gut content screened for DNA of both aphid species using multiplex PCR. Comparing detection frequencies of DNA from living and dead prey, we found that all predators frequently consumed dead aphids, except for the lycosid spider Trochosa ruricola which exclusively fed on S. avenae . In contrast, in the tetragnathid spider Pachygnatha degeeri , supposed to feed mainly on living prey, detection rates of R. padi DNA were high. Our findings support previous assumptions that many generalist predators are facultative scavengers. This needs to be considered for the interpretation of field-derived data using approaches of gut content analyses as well as when assessing predators’ pest control potential.

Impact of abiotic factors on predator-prey interactions: DNA-based gut content analysis in a microcosm experiment

The effects of predators on prey populations can be modified by a number of abiotic factors. Here, we investigated the combined and separate effects of rain and ground-dwelling predators on aphid populations in a microcosm experiment lasting for 21 days, using PCR to analyse the gut content of the predators. Rain significantly dislodged aphids from shoots and ears by 57% and 25%, respectively. The gut content analysis showed that more predators consumed aphids in the rain treatment than without rain, indicating higher availability of aphids to ground-dwelling predators after rain. However, no synergistic effects of rain and ground-dwelling predators on aphid population development could be demonstrated. Rain alone significantly decreased aphid populations by 27%, suggesting that this is a significant mortality factor. Predators alone had no significant effect on aphid numbers, but the gut content analyses showed aphid consumption also in the no-rain treatments, indicating that aphids were available to the predators on the soil surface even without rain. Our results suggest that weather conditions such as rain can modify predator-prey interactions in the field. Employing PCR-based predator gut content analyses proved to be useful as trophic links could be directly verified.

Earthworm primers for DNA-based gut content analysis and their cross-reactivity in a multi-species system

Specific primers can be used in polymerase chain reactions (PCR) to amplify prey DNA from the gut content of generalist predators at high specificity and sensitivity. A prerequisite for applying this approach to field studies, however, is to confirm that primers are actually targeting specific prey species or prey groups and do not produce false positive results by amplifying DNA either from predator species or from the wide range of potential alternative prey found under natural conditions. Here, we report on a new group-specific primer pair for earthworms designed from cytochrome oxidase c subunit 1 (COI) sequences of 11 earthworm species found in Central Europe that can be used to detect consumption of earthworms by invertebrate predators. Besides inter-specific also considerable intra-specific variation was found for COI sequences among most of the earthworm species. We, therefore, combined a universal forward primer with an earthworm-specific reverse primer which amplified a 523 bp product from all 11 species tested. Earthworm DNA amplification was also successful in the presence of excess DNA of a predator species. The primer pair was tested against 82 non-target invertebrate species commonly found in the same habitats, including potential prey for generalist predators and predators themselves. The earthworm primer was highly specific: only one of the non-target species showed a product of similar length as the earthworms, whereas PCR with 12 non-target species produced amplicons whose length differed from that of earthworms. We conclude that the new primer will be a useful tool to investigate the role earthworms play as a food resource in soil food-webs. Moreover, we suggest that future studies utilizing DNA-based approaches for prey detection should select non-target species for cross-reactivity tests according to their abundance and importance rather than choosing representatives of taxonomic units; this will help validate the results achieved using species- or group-specific primers and guarantee their meaningful ecological interpretation.