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

Molecular analysis of predation, through polymerase chain reaction amplification of prey remains within the faeces or digestive systems of predators, is a rapidly growing field, impeded by a lack of readily accessible advice on best practice. Here, we review the techniques used to date and provide guidelines accessible to those new to this field or from a different molecular biology background. Optimization begins with field collection, sample preservation, predator dissection and DNA extraction techniques, all designed to ensure good quality, uncontaminated DNA from semidigested samples. The advantages of nuclear vs. mitochondrial DNA as primer targets are reviewed, along with choice of genes and advice on primer design to maximize specificity and detection periods following ingestion of the prey by the predators. Primer and assay optimization are discussed, including cross-amplification tests and calibratory feeding experiments. Once primers have been made, the screening of field samples must guard against (through appropriate controls) cross contamination. Multiplex polymerase chain reactions provide a means of screening for many different species simultaneously. We discuss visualization of amplicons on gels, with and without incorporation of fluorescent primers. In more specialized areas, we examine the utility of temperature and denaturing gradient gel electrophoresis to examine responses of predators to prey diversity, and review the potential of quantitative polymerase chain reaction systems to quantify predation. Alternative routes by which prey DNA might get into the guts of a predator (scavenging, secondary predation) are highlighted. We look ahead to new technologies, including microarrays and pyrosequencing, which might one day be applied to this field.     

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.     

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

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

Removing external DNA contamination from arthropod predators destined for molecular gut-content analysis

Ecological research requires large samples for statistical validity, typically hundreds or thousands of individuals, which are most efficiently gathered by mass-collecting techniques. For the study of interspecific interactions, molecular gut-content analysis enables detection of arthropod predation with minimal disruption of community interactions. Field experiments have demonstrated that standard mass-collection methods, such as sweep netting, vacuum sampling and foliage beating, sometimes lead to contamination of predators with nontarget DNA, thereby compromising resultant gut-content data. We deliberately contaminated immature Coleomegilla maculata and Podisus maculiventris that had been fed larvae of Leptinotarsa decemlineata by topically applying homogenate of the alternate prey Leptinotarsa juncta. We then attempted to remove contaminating DNA by washing in ethanol or bleach. A 40-min wash with end-over-end rotation in 80% EtOH did not reliably reduce external DNA contamination. Identical treatment with 2.5% commercial bleach removed most externally contaminating DNA without affecting the detectability of the target prey DNA in the gut. Use of this bleaching protocol, perhaps with minor modifications tailored to different predator-prey systems, should reliably eliminate external DNA contamination, thereby alleviating concerns about this possible source of cross-contamination for mass-collected arthropod predators destined for molecular gut-content analysis.     

PCR-based gut content analysis of insect predators: using ribosomal ITS-1 fragments from prey to estimate predation frequency

We used polymerase chain reaction to determine whether Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) DNA was present in the guts of larvae and adult males and females of the generalist predator Coleomegilla maculata De Geer (Coleoptera: Coccinellidae). The predators were fed Ostrinia nubilalis egg masses and allowed to digest at either 20 degrees C or 27 degrees C for time spans ranging from 0 to 12 h. Four primer pairs, specific for O. nubilalis were developed, using a nuclear ribosomal RNA sequence including part of the 18S gene, the complete internal transcribed spacer (ITS-1) region and part of the 5.8S gene. These primers amplified four sequences that were 492, 369, 256 and 150 base pairs long. We found a significant negative effect of time since feeding on the number of bands that could be detected. The shortest fragment was detected for the longest time after feeding (up to 12 h). We found no effect of predator weight, sex, developmental stage, or meal size on the time course over which bands of varying lengths could be detected.     

Unnecessary roughness? Testing the hypothesis that predators destined for molecular gut‐content analysis must be hand‐collected to avoid cross‐contamination

Molecular gut‐content analysis enables detection of arthropod predation with minimal disruption of ecosystem processes. Mass‐collection methods, such as sweep‐netting, vacuum sampling and foliage beating, could lead to regurgitation or rupturing of predators along with uneaten prey, thereby contaminating specimens and compromising resultant gut‐content data. Proponents of this ‘cross‐contamination hypothesis’ advocate hand‐collection as the best way to avoid cross‐contamination. However, hand‐collection is inefficient when large samples are needed, as with most ecological research. We tested the cross‐contamination hypothesis by setting out onto potato plants immature Coleomegilla maculata and Podisus maculiventris that had been fed larvae of either Leptinotarsa decemlineata or Leptinotarsa juncta, or unfed individuals of these predator species along with L. decemlineata larvae. The animals were then immediately re‐collected, either by knocking them vigorously off the plants onto a beat cloth and capturing them en masse with an aspirator (‘rough’ treatment) or by hand‐searching and collection with a brush (‘best practice’). Collected predators were transferred in the field to individual vials of chilled ethanol and subsequently assayed by PCR for fragments of cytochrome oxidase I of L. decemlineata and L. juncta. Ten to 39 per cent of re‐collected fed predators tested positive by PCR for DNA of both Leptinotarsa species, and 14–38% of re‐collected unfed predators contained L. decemlineata DNA. Overall levels of cross‐contamination in the rough (31%) and best‐practice (11%) samples were statistically different and supported the cross‐contamination hypothesis. A pilot study on eliminating external DNA contamination with bleach prior to DNA extraction and amplification gave promising results.     

Pollen beetles are consumed by ground‐ and foliage‐dwelling spiders in winter oilseed rape

Pollen beetles, Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae), are major pests in oilseed rape (OSR), Brassica napus L. (Brassicaceae). Among the predator species in the generalist predator complex present in OSR fields, wolf spiders (Araneae: Lycosidae) are found on the ground and cobweb spiders (Araneae: Theridiidae) build webs in the foliage. Here we study the incidence of predation of pollen beetles by these two spider groups using DNA‐based molecular analysis. Wolf spiders of the genus Pardosa and the cobweb spider, Theridion impressum L. Koch, were each collected in three winter OSR fields over a period of about 3 weeks. Pollen beetle densities as well as the occurrence of predators and alternative prey were monitored. In total, 13.8% of the collected Pardosa spp. tested positive for pollen beetle DNA in the PCR analyses, whereas 51.7%T. impressum were positive. The likelihood of detecting pollen beetle DNA in the gut contents of both spider groups was positively related to pollen beetle larval density. The implications of these results for conservation biological control and future studies of food webs in OSR are discussed.     

Molecular detection of predation by soil micro-arthropods on nematodes

The relative importance of the factors driving change in the population dynamics of nematodes in the soil is almost completely unknown. Top-down control by micro-arthropod predators may have a significant impact on nematode population dynamics. We report experiments showing that mites and Collembola were capable of reducing nematode numbers in the laboratory and were feeding on a targeted nematode species in the field. A PCR-based approach was developed for the detection of predation on three species of slug- and insect-pathogenic nematodes: Phasmarhabditis hermaphrodita, Heterorhabditis megidis and Steinernema feltiae. The collembolan Folsomia candida and the mesostigmatid mite Stratiolaelaps miles were employed as model predators to calibrate post-ingestion prey DNA detection times. Fragments of cytochrome oxidase I (COI) mtDNA were sequenced and species-specific primers were designed, amplifying 154-, 154- and 203-bp fragments for each of the nematode species. Detection times for nematode DNA within the guts of Collembola were longer than in mites, with half-lives (50% of samples testing positive) of 08.75 h and 05.03 h, respectively. F. candida significantly reduced numbers of the nematode H. megidis, with rates of predation of approximately 0.4 nematode infective juveniles per collembolan per hour over 10 h. Four taxa of field-caught micro-arthropod that had been exposed to the nematode P. hermaphrodita for a period of 12 h were analysed and significant numbers of three taxa tested positive. This is the first application of PCR techniques for the study of nematophagy and the first time these techniques have been used to measure predation on nematodes in the field.     

A comparative analysis of spider prey spectra analyzed through the next‐generation sequencing of individual and mixed DNA samples

As one of the most abundant predators of insects in terrestrial ecosystems, spiders have long received much attention from agricultural scientists and ecologists. Do spiders have a certain controlling effect on the main insect pests of concern in farmland ecosystems? Answering this question requires us to fully understand the prey spectrum of spiders. Next‐generation sequencing (NGS) has been successfully employed to analyze spider prey spectra. However, the high sequencing costs make it difficult to analyze the prey spectrum of various spider species with large samples in a given farmland ecosystem. We performed a comparative analysis of the prey spectra of Ovia alboannulata (Araneae, Lycosidae) using NGS with individual and mixed DNA samples to demonstrate which treatment was better for determining the spider prey spectra in the field. We collected spider individuals from tea plantations, and two treatments were then carried out: (1) The DNA was extracted from the spiders individually and then sequenced separately (DESISS) and (2) the DNA was extracted from the spiders individually and then mixed and sequenced (DESIMS). The results showed that the number of prey families obtained by the DESISS treatment was approximately twice that obtained by the DESIMS treatment. Therefore, the DESIMS treatment greatly underestimated the prey composition of the spiders, although its sequencing costs were obviously lower. However, the relative abundance of prey sequences detected in the two treatments was slightly different only at the family level. Therefore, we concluded that if our purpose were to obtain the most accurate prey spectrum of the spiders, the DESISS treatment would be the best choice. However, if our purpose were to obtain only the relative abundance of prey sequences of the spiders, the DESIMS treatment would also be an option. The present study provides an important reference for choosing applicable methods to analyze the prey spectra and food web compositions of animal in ecosystems.     

Gut content analysis to study predatory efficacy of Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) by molecular methods

Nesidiocoris tenuis is considered to make significant contributions to the control of greenhouse pests such as whiteflies, thrips, leafminers, lepidopterans, and spider mites, although there is little information based on direct observation of the predation of N. tenuis on these target pests. We developed a method to perform gut content analysis of N. tenuis based on DNA in which specific PCR primers were designed to detect the DNA of target pests. By means of gut‐content analysis, we found that the percentage of N. tenuis preying on Bemisia tabaci and Thrips palmi was approximately 40% in the field.     

Tracking predation of subterranean pests: digestion of corn rootworm DNA by a generalist mite

qPCR is a useful tool for understanding predator–prey relationships. We investigated rootworm DNA digestion by male and female predatory mites. Males and females initially consumed comparable amounts of DNA, which was digested at similar rates. Field-collected mites need to be preserved quickly for best qPCR results.     

Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples

Molecular techniques have become an important tool to empirically assess feeding interactions. The increased usage of next‐generation sequencing approaches has stressed the need of fast DNA extraction that does not compromise DNA quality. Dietary samples here pose a particular challenge, as these demand high‐quality DNA extraction procedures for obtaining the minute quantities of short‐fragmented food DNA. Automatic high‐throughput procedures significantly decrease time and costs and allow for standardization of extracting total DNA. However, these approaches have not yet been evaluated for dietary samples. We tested the efficiency of an automatic DNA extraction platform and a traditional CTAB protocol, employing a variety of dietary samples including invertebrate whole‐body extracts as well as invertebrate and vertebrate gut content samples and feces. Extraction efficacy was quantified using the proportions of successful PCR amplifications of both total and prey DNA, and cost was estimated in terms of time and material expense. For extraction of total DNA, the automated platform performed better for both invertebrate and vertebrate samples. This was also true for prey detection in vertebrate samples. For the dietary analysis in invertebrates, there is still room for improvement when using the high‐throughput system for optimal DNA yields. Overall, the automated DNA extraction system turned out as a promising alternative to labor‐intensive, low‐throughput manual extraction methods such as CTAB. It is opening up the opportunity for an extensive use of this cost‐efficient and innovative methodology at low contamination risk also in trophic ecology.     

A comparative analysis of PCR-based detection methods for avaian malaria

Here, 4 polymerase chain reaction (PCR) assays are compared to test for the presence of avian malaria, including both the Plasmodium and Haemoproteus genera, in 29 different species of African rainforest birds. Two of these PCR assays use primer sets that amplify fragments of the cytochrome b (cyt b) gene of Plasmodium; the other 2 target the 18S ribosomal subunit gene. These PCR assays were performed using genomic DNA extracted from blood and subsequently compared with the results obtained by microscopic examination of blood smears taken from the same individuals. The 2 primer sets amplifying the cyt b gene were found to perform more reliably than those that target the 18S rRNA gene and yielded a substantial number of positive samples that were undetected by blood smear analysis. Of all the individuals screened by PCR, 40% tested positive for avian malaria, whereas 27% tested positive by blood smear analysis. Although sequence variation in the parasites may prohibit the specific alignment of primers and the subsequent PCR amplification of some individuals, PCR, once optimized, is faster, cheaper, and more reliable than blood smear analysis for large-scale screening.     

Estimating predation rates from molecular gut content analysis

Several methods have been published to estimate per capita predation rates from molecular gut content analysis relying on intuitive understanding of predation, but none have been formally derived. We provide a theoretical framework for estimating predation rates to identify an accurate method and lay bare its assumptions. Per capita predation can be estimated by multiplying the prey decay rate and the prey quantity in the predators. This assumes that variation in per capita predation rate is approximately normally distributed, prey decay occurs exponentially, and predation is in steady state. We described several ways to estimate steady state predation, including using only qualitative presence-absence data to estimate the decay rate and in addition, we provided a method for estimating per capita predation rate when predation is not in steady state. We used previously published data on aphid consumption by a ladybird beetle in a feeding trial to calculate the predation rate and compare published methods with this theoretically derived method. The estimated predation rate (3.29 ± 0.27 aphids/h) using our derived method was not significantly different from the actual predation rate, 3.11 aphids/h. In contrast, previously published methods were less accurate, underestimating the predation rate (0.33 ± 0.02 to 1.66 ± 0.8 aphids/h) or overestimating it (3.64 ± 0.30 aphids/h). In summary, we provide methods to estimate predation rates even when variation in predation rates is not exactly normally distributed and not in steady state and demonstrate that the prey decay rate, and not the prey detection period, is required.     

Distinct feeding strategies of generalist and specialist spiders

1. Feeding behaviour of generalist and specialist predators is determined by a variety of trophic adaptations. Specialised prey‐capture adaptations allow specialists to catch relatively large prey on a regular basis. As a result, specialists might be adapted to exploit each item of prey more thoroughly than do generalists. 2. It was expected that obligatory specialist cursorial spiders would feed less frequently than generalists but for a longer time and, thus, that their foraging pause would be longer. First, the feeding frequencies of three generalist spider species (Cybaeodamus taim, Harpactea hombergi, Hersiliola sternbergsi) were compared with those three phylogenetically related specialist species: myrmecophagous Zodarion rubidum, and araneophagous Nops aff. variabilis and Palpimanus orientalis. 3. Generalists captured more prey, exploited each item of prey for a significantly shorter time, and had a shorter foraging pause than was the case for specialists. Generalists also gained significantly less relative amount of prey mass than did specialists. 4. Second, the study compared the prey DNA degradation rate in the gut of generalists and specialists by means of PCR. The degradation rate was not significantly different between specialists and generalists: the detectability half‐life was estimated to exist for 14.3 days after feeding. 5. This study shows that the feeding strategies of cursorial generalist and obligatory specialist spiders are different. Obligatory specialists have evolved a feeding strategy that is based on thorough exploitation of a few large prey, whereas generalists have evolved a strategy that is based on short exploitation of multiple small items of prey.     

群落二维格局分析的两种方法

群落二维空间格局研究能够更好地揭示群落的特征,但在分析方法上有较大的困难。用垂直相交的两条样带在两个方向上同时取样的二维取样法,获得数据,用DCA排序与格局分析相结合的方法,得到群落不同格局规模斑块的长、宽及面积,实现二维格局研究。在亚高山草甸群落格局研究中应用表明,两个方法均是有效的。     

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.     

Common methods for cytosine methylation analysis in DNA

The review considers the methods most commonly used to detect DNA methylation, their advantages, potential limitations, and selection for various purposes. A detailed protocol is described for bisulfite treatment, which is used as a preliminary step in the majority of DNA methylation assays.     

A DNA marker to identify predation of Plutella xylostella (Lep., Plutellidae) by Nabis kinbergii (Hem., Nabidae) and Lycosa sp. (Aranaea, Lycosidae)

Abstract:  Predators are important biotic factors in the population dynamics of the diamondback moth, Plutella xylostella . A specific DNA marker was developed to detect P. xylostella in the gut contents of two polyphagous predators, Nabis kinbergii and Lycosa sp. A distinct 275-bp product was amplified by polymerase chain reaction (PCR) from the internal transcribed spacer (ITS-1) of the ribosomal gene of P. xylostella , but not from 11 other arthropod species collected from Brassica fields in South Australia. Fortuitously, the primer set could also amplify DNA products from two species and three varieties of Brassica plants, with the fragment size about 600 bp. When N. kinbergii was analysed after feeding a single fourth instar P. xylostella , 67% of individuals were positive with the 275-bp PCR product up to 16 h after feeding. Likewise, the PCR product was detected in 80% individuals of Lycosa sp. up to 72 h after feeding on a single fourth instar P. xylostella larva. Initial tests of samples collected from the field showed that the predation incidences for both N. kinbergii and Lycosa sp. determined by the 275-bp fragment corresponded to the density of P. xylostella in the field.