Taxon-specific PCR for DNA barcoding arthropod prey in bat faeces

The application of DNA barcoding to dietary studies allows prey taxa to be identified in the absence of morphological evidence and permits a greater resolution of prey identity than is possible through direct examination of faecal material. For insectivorous bats, which typically eat a great diversity of prey and which chew and digest their prey thoroughly, DNA-based approaches to diet analysis may provide the only means of assessing the range and diversity of prey within faeces. Here, we investigated the effectiveness of DNA barcoding in determining the diets of bat species that specialize in eating different taxa of arthropod prey. We designed and tested a novel taxon-specific primer set and examined the performance of short barcode sequences in resolving prey species. We recovered prey DNA from all faecal samples and subsequent cloning and sequencing of PCR products, followed by a comparison of sequences to a reference database, provided species-level identifications for 149/207 (72%) clones. We detected a phylogenetically broad range of prey while completely avoiding detection of nontarget groups. In total, 37 unique prey taxa were identified from 15 faecal samples. A comparison of DNA data with parallel morphological analyses revealed a close correlation between the two methods. However, the sensitivity and taxonomic resolution of the DNA method were far superior. The methodology developed here provides new opportunities for the study of bat diets and will be of great benefit to the conservation of these ecologically important predators.     

Proportion of prey consumed can be determined from faecal DNA using real-time PCR

Reconstructing the diets of pinnipeds by visually identifying prey remains recovered in faecal samples is challenging because of differences in digestion and passage rates of hard parts. Analysing the soft-matrix of faecal material using DNA-based techniques is an alternative means to identify prey species consumed, but published techniques are largely nonquantitative, which limits their usefulness for some applications. We further developed and validated a real-time PCR technique using species-specific mitochondrial DNA primers to quantify the proportion of prey in the diets of Steller sea lions (Eumetopias jubatus), a pinniped species thought to be facing significant diet related challenges in the North Pacific. We first demonstrated that the proportions of prey tissue DNA in mixtures of DNA isolated from four prey species could be estimated within a margin of ～ 12% of the percent in the mix. These prey species included herring Clupea palasii, eulachon Thaleichthyes pacificus, squid Loligo opalescens and rosethorn rockfish Sebastes helvomaculatus. We then applied real-time PCR to DNA extracted from faecal samples obtained from Steller sea lions in captivity that were fed 11 different combinations of herring, eulachon, squid and Pacific ocean perch rockfish (Sebastes alutus), ranging from 7% to 75% contributions per meal (by wet weight). The difference between the average percentage estimated by real-time PCR and the percentage of prey consumed was generally <12% for all diets fed. Our findings indicate that real-time PCR of faecal DNA can detect the approximate relative quantity of prey consumed for complex diets and prey species, including cephalopods and fish.     

Sensitive detection of FGFR3 mutations in bladder cancer and urine sediments by peptide nucleic acid-mediated real-time PCR clamping

Somatic mutations of the fibroblast growth factor receptor 3 (FGFR3) gene were detected by peptide nucleic acid (PNA)-mediated real-time PCR clamping. Mutation was detected in negative control containing only wild-type DNA due to a misincorporation of dNTPs to PNA binding sites when the amount of template DNA was decreased to 1 ng. Thus, the amount of template DNA was critical determinant of the assay sensitivity in PNA-mediated PCR clamping. Assay conditions were optimized to detect FGFR3 mutations in exons 7, 10, and 15, at a concentration of more than 1% mutated DNA using 50 ng of genomic DNA as the template. Mutations were detected in 12 of 13 (92.3%) tumor tissues and 11 of 13 (84.6%) urine samples from patients with superficial bladder cancer, while no mutations were detected in tissues and/or urine samples from patients with muscle-invasive bladder cancer or chronic cystitis.     

核酸技术在立枯丝核菌分类与生态学研究中的应用

立枯丝核菌是一种传播广、危害大的土传病原菌,对其展开的研究已涉及各个方面并逐渐深入,近几年借助于核酸技术对其所展开的研究更成为热点。对核酸技术在立枯丝核菌的分类学研究(主要包括G Cmol%含量测定、核酸杂交、各种DNA指纹技术、特异PCR扩增、测序DNA的序列分析)、监测群体动态变化的生态学研究(实时荧光PCR技术)中的应用作一简要的介绍。     

A century of Chinook salmon consumption by marine mammal predators in the Northeast Pacific Ocean

As many marine mammal populations have increased following bans on their harvest, there has been a growing need to understand potential impacts of these population changes on coastal marine ecosystems. Quantifying consumption of prey species, such as fish, is particularly important when those same prey are also targeted by commercial fisheries. Estimating the impact of marine mammal predators on prey fish depends upon knowledge of marine mammal diet composition; scientific advances over the last century have improved understanding of diets but have also led to inconsistent methods that challenge attempts at synthesis and comparison. Meta-analysis techniques offer the opportunity to overcome such challenges, yet have not been widely applied to synthesize marine mammal diets over space and time. As a case study, we focus on synthesizing diet studies of Chinook (king) salmon (Oncorhynchus tshawytscha) by four species of marine mammal predators in the Northeast Pacific Ocean: Steller sea lions (Eumetopias jubatus), California sea lions (Zalophus californianus), harbor seals (Phoca vitulina), and killer whales (Orcinus orca). We also highlight several simple meta-analyses for which these types of diet databases may be employed. Our assembled database consists of > 330 records, spanning more than 100 years. Results indicate that the frequency of occurrence of Chinook salmon in killer whale studies is high (63%) relative to pinniped studies (< 10%). They also suggest a strong increasing ability to discriminate Chinook salmon from other salmonids, which we attribute to switches in diet studies from lethal or observational sampling toward molecular methods (DNA, fatty acids). Our database and analysis code are published as supplementary material, which we hope will be useful for other researchers and will inspire more of these syntheses.     

The apparent diet of predators and biases due to different handling times of their prey

Summary In order to estimate the true diet of predators, the prey of a number of predators is recorded at one time. Such sampling underestimates the true diet during a period of time. Where handling times of different types of prey are very different, these estimates will be biased, because prey that take a relatively long time to be eaten will be overestimated. We examined a rocky intertidal predatorprey system and demonstrated the existence of such bias. A number of hypothetical correlates of the bias were also investigated. As anticipated, variations in handling times were a major factor, but neither taxonomic affinity nor absolute size of the prey could predict the degree of underestimation in the true diet for any given type of prey. A previously described correction for this type of bias was tested, but found to be unsatisfactory. We suggest that it was too insensitive to variability in handling times.A simple computer model incorporating differences among prey in their handling times was also unable to predict the bias, but did indicate that non-random selection of prey was occurring.We concluded that where such biases are likely to occur, information on the handling times of different prey and/or accurate estimates of the true diets of the predators are essential for the predatory interaction to be interpreted properly. These results were discussed in relation to published accounts of diets of predators in rocky intertidal habitats. Many studies have not presented data on handling times of prey in the field, and the magnitude and importance of potential biases in these studies are therefore difficult to assess.     

Investigation on Natural Diets of Larval Marine Animals Using Peptide Nucleic Acid-Directed Polymerase Chain Reaction Clamping

The stomach contents of the larvae of marine animals are usually very small in quantity and amorphous, especially in invertebrates, making morphological methods of identification very difficult. Nucleotide sequence analysis using polymerase chain reaction (PCR) is a likely approach, but the large quantity of larval (host) DNA present may mask subtle signals from the prey genome. We have adopted peptide nucleic acid (PNA)-directed PCR clamping to selectively inhibit amplification of host DNA for this purpose. The Japanese spiny lobster (Panulirus japonicus) and eel (Anguilla japonica) were used as model host and prey organisms, respectively. A lobster-specific PNA oligomer (20 bases) was designed to anneal to the sequence at the junction of the 18 S rDNA gene and the internal transcribed spacer 1 (ITS1) of the lobster. PCR using eukaryote universal primers for amplifying the ITS1 region used in conjunction with the lobster-specific PNA on a mixed DNA template of lobster and eel demonstrated successful inhibition of lobster ITS1 amplification while allowing efficient amplification of eel ITS1. This method was then applied to wild-caught lobster larvae of P. japonicus and P. longipes bispinosus collected around Ryukyu Archipelago, Japan. ITS1 sequences of a wide variety of animals (Ctenophora, Cnidaria, Crustacea, Teleostei, Mollusca, and Chaetognatha) were detected.     

Should fatty acid signature proportions sum to 1 for diet estimation?

Knowledge of predator diets, including how diets might change through time or differ among predators, provides essential insights into their ecology. Diet estimation therefore remains an active area of research within quantitative ecology. Quantitative fatty acid signature analysis (QFASA) is an increasingly common method of diet estimation. QFASA is based on a data library of prey signatures, which are vectors of proportions summarizing the fatty acid composition of lipids, and diet is estimated as the mixture of prey signatures that most closely approximates a predator’s signature. Diets are typically estimated using proportions from a subset of all fatty acids that are known to be solely or largely influenced by diet. Given the subset of fatty acids selected, the current practice is to scale their proportions to sum to 1.0. However, scaling signature proportions has the potential to distort the structural relationships within a prey library and between predators and prey. To investigate that possibility, we compared the practice of scaling proportions with two alternatives and found that the traditional scaling can meaningfully bias diet estimators under some conditions. Two aspects of the prey types that contributed to a predator’s diet influenced the magnitude of the bias: the degree to which the sums of unscaled proportions differed among prey types and the identifiability of prey types within the prey library. We caution investigators against the routine scaling of signature proportions in QFASA.     

Diet choice in an omnivorous salt-marsh crab: different food types, body size, and habitat complexity

Studies of diet choice by omnivores have the potential to form conceptual links between studies of diet choice by herbivores, frugivores, detritivores, and predators. We examined diet choice in the omnivorous salt marsh crab Armases cinereum (=Sesarma cinereum (Grapsidae)) in a series of laboratory experiments. Armases is sexually dimorphic, with larger males having relatively larger claws than females. In a growth experiment, an invertebrate diet supported better growth than any other single diet; however, growth also occurred on single diets of mud, leaf litter or fresh leaves. Mixed diets provided the best growth. If alternative foods were available, consumption of leaf litter and fresh leaves decreased, but these items were not dropped from the diet completely. In contrast, consumption of invertebrate prey was not affected by the availability of alternative foods. In a predation experiment, crustacean prey (an amphipod and an isopod) were more vulnerable to predation by Armases than were two small gastropod species. Only large male Armases were able to consume large numbers of gastropods. Environmental structure (plant litter or litter mimics) reduced predation rates, especially on crustaceans, which actively utilized the structure to hide from predators. Armases consumes a mixed diet because several factors (prey physical defenses, avoidance behavior of prey, growth benefits of a mixed diet) favor omnivory over a specialized diet. Similar factors may promote minor amounts of “omnivory” by species generally considered to be herbivores, frugivores, detritivores, and predators.     

Diet of intraguild predators affects antipredator behavior in intraguild prey

In two-predator, one-prey systems with intraguild predationand patchily distributed prey, the intraguild prey may facea choice between prey patches with and without intraguild predators.To minimize falling victim to intraguild predation, intraguildprey are expected to perceive cues specifically associated withthe presence of intraguild predators. We investigate whetherintraguild prey avoided intraguild predators and which cuestriggered this behavior in a system composed of plant-inhabitingarthropods. We found that intraguild prey recognized intraguildpredators from a distance, based on their diet: they avoidedodors of intraguild predators that had consumed shared preybut did not avoid odors of intraguild predators that had fedon other diets, including a diet of conspecifics. When intraguildprey were foraging on a patch, detection of intraguild predatorsled to longer periods of immobility and to fewer captures ofthe shared prey. However, intraguild predators that were eitherstarved or had previously consumed intraguild prey posed a higherrisk to intraguild prey than did intraguild predators that hadconsumed the shared prey. We conclude that the cues used byintraguild prey to avoid intraguild predators are associatedwith the circumstances under which they encounter intraguildpredators in the field and not to different degrees of danger.     

Assessing morphological and DNA-based diet analysis techniques in a generalist predator, the arrow squid Nototodarus gouldi

Establishing the diets of marine generalist consumers is difficult, with most studies limited to the use of morphological methods for prey identification. Such analyses rely on the preservation of diagnostic hard parts, which can limit taxonomic resolution and introduce biases. DNA-based analyses provide a method to assess the diets of marine species, potentially overcoming many of the limitations introduced by other techniques. This study compared the effectiveness of morphological and DNA-based analysis for determining the diet of a free-ranging generalist predator, the arrow squid (Nototodarus gouldi). A combined approach was more effective than using either of the methods in isolation. Nineteen unique prey taxa were identified, of which six were found by both methods, 10 were only detected using DNA and three were only identified using morphological methods. Morphological techniques only found 50% of the total number of identifiable prey taxa, whereas DNA-based techniques found 84%. This study highlights the benefits of using a combination of techniques to detect and identify prey of generalist marine consumers.     

Weak responses to dietary enrichment in a specialized aphid predator

Some ladybeetles are specialist predators of aphids, coccids or other prey, although they often eat a variety of species from their focal prey taxon. In addition, the diet is often supplemented with alternative prey. How larvae of the aphidophagous Coccinella septempunctata L. utilize a non‐aphid alternative prey (fruit‐fly larvae Drosophila melonogaster Meigen) is compared with adequate (i.e. high‐quality) aphid prey provided alone (monotypic diet) or in mixed diets. The alternative prey are presented either nutrient‐enriched (i.e. raised on dog food supplemented medium) or not (raised on pure medium). Ladybird performance (survival, growth and development) is poor on the pure fly larvae diets, and also reduced when given mixed diets compared with the pure aphid diet. Nutrient enrichment of the fly larvae has no positive effects. The physiological background for the differences in food value, as indicated by performance in life‐history parameters, is a strong pre‐ingestive effect (i.e. reduced consumption of fly larvae compared with aphids) and a post‐ingestive effect (i.e. reduced utilization of assimilated larval fly tissue), whereas the assimilation efficiency of the consumed fly larvae is as high as that of aphids. The results show a physiological trade‐off resulting from prey specialization that reduces the possibility of utilizing alternative prey when the availability of aphids is scarce. Connected with this is a high robustness against variation in prey nutrient diversity and composition; the ladybird shows little positive response to dietary mixing (i.e. neither mixing of adequate aphids, nor of aphids and alternative prey) or to nutrient enrichment of prey. This contrasts with the results from generalist predators (spiders), where similar treatments lead to strong effects on life‐history parameters.     

Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology

During the past ten years, the DNA mimic peptide nucleic acid has inspired the development of a variety of hybridisation-based methods for detection, quantification, purification and characterisation of nucleic acids. Most of these methods have taken advantage of the very favourable DNA and RNA hybridisation properties of peptide nucleic acids combined with the unique properties and opportunities offered by peptide chemistry. Within the past year, significant progress in in situ hybridisation technology has been achieved, which has resulted, in particular, in reliable and sensitive methods for detection of bacteria in clinical samples, as well as in environmental samples. Furthermore, applications of the polymerase chain reaction clamping method have been expanded, and novel ways of exploiting complexes of peptide nucleic acids with double-stranded DNA, such as double duplex invasion complexes and PD loops, have been developed.     

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.     

DNA as a Dietary Biomarker in Antarctic Krill, Euphausia superba

The diet of Antarctic krill (Euphausia superba) has been studied using a variety of techniques, but current methods still suffer from problems that are difficult to solve. This study examined an alternative approach utilizing DNA as a prey biomarker. Methods were developed for the preservation, extraction, and identification of prey DNA from krill collected in the field. Group-specific polymerase chain reaction (PCR) was used to amplify diatom prey (Phylum: Bacillariophyta) and the results from DNA clone libraries were compared with microscopic diet analysis. DNA analysis was superior to microscopy for prey detection. However, differences in prey relative abundance estimates between the two techniques suggested some bias in the DNA-based estimates. Quantification showed that large amounts of prey DNA had been successfully preserved and extracted. Overall the results suggest that the application of DNA-based diet analysis to krill warrants further investigation, particularly for prey that are difficult to study using other methods.     

Detecting frogs as prey in the diets of introduced mammals: a comparison between morphological and DNA‐based diet analyses

Amphibians are currently the most threatened group of vertebrates worldwide, and introduced fauna play a major role in their decline. The control of introduced predators to protect endangered species is often based on predation rates derived from diet studies of predators, but prey detection probabilities using different techniques are variable. We measured the detectability of frogs as prey, using morphological and DNA‐based diet analyses, in the stomachs and faeces of four mammal species that have been introduced to many areas of the world. Frogs (Litoria raniformis) were fed to rats (Rattus norvegicus and R. rattus), mice (Mus musculus) and hedgehogs (Erinaceus europaeus). DNA‐based analysis outperformed morphological analysis, increasing the prey detection rate from 2% to 70% in stomachs and from 0% to 53% in faeces. In most cases, utilizing either stomachs or faeces did not affect the success of prey DNA detection; however, using faeces extended the detectability half‐life from 7 to 21 h. This study is the first to measure prey DNA detection periods in mammalian stomachs, and the first to compare prey DNA detection periods in the stomachs and faeces of vertebrates. The results indicate that DNA‐based diet analysis provides a more reliable approach for detecting amphibians as prey and has the potential to be used to estimate the rate of predation by introduced mammals on endangered amphibians.     

Chemical Predator Inspection in a Characin Fish (Hemigrammus erythrozonus, Characidae, Ostariophysi): The Effects of Mixed Predator Diets

Many prey organisms will approach (inspect) potential predators, primarily to assess local risk of predation. It has been demonstrated that Ostariphysan prey fishes can detect conspecific alarm pheromones in the diet of potential predators and use this chemical information to reduce their risk of predation while still gaining significant benefits associated with predator inspection. We conducted the current study to examine the possible effects of mixed diets on the use of these chemical predator diet cues during inspection visits. Shoals of four glowlight tetras ( Hemigrammus erythrozonus ) were exposed to Jack Dempsey cichlids ( Cichlasoma octofaciatum ) which had been fed diets consisting of: 100% tetras (with alarm pheromone); 75% tetra, 25% swordtail ( Xiphophorus helleri , which lack a recognizable alarm pheromone); 25% tetra, 75% swordtail; or 100% swordtails. Tetras significantly increased their anti-predator behaviour in response to predators fed 100% tetra or the two mixed predator diets, but not when exposed to predators fed a 100% swordtail diet. Likewise, we observed significant differences in inspection behaviour. Tetras took longer to initiate an inspection, inspected in smaller groups and directed a greater proportion of inspection visits towards the tail region of the predator when it had been fed 100% tetra or either of the two mixed prey diets. We found no significant differences in either anti-predator or inspection behaviour among the three diet treatments containing tetras. These data strongly suggest that glowlight tetras are capable of detecting relatively small amounts of conspecific alarm pheromone in the diet of potential predators and that they modify their behaviour based on the presence or absence of these cues.     

捕食性天敌昆虫控害作用定量评价方法

天敌昆虫田间捕食作用与能力的研究,通常采用田间种群数量调查与分析的方法或笼罩试验来评价其控制作用,但往往与实际情况不符,尤其是对可捕食多种猎物的非专食性天敌,很难说明其在田间对某一特定猎物的控制作用究竟有多大,而分子生物学技术为捕食者-猎物关系评价研究提供了新途径。本文系统介绍了基于种特异性SS-PCR标记技术和TaqMan-MGB实时荧光定量PCR技术的捕食性天敌控害作用定量评价方法及其优缺点,旨在为害虫天敌的种类筛选及其保护利用提供技术指导。     

A resolution of the paradox of enrichment

Theoretical studies have shown a paradoxical destabilizing response of predator-prey ecosystems to enrichment, but there is the gap between the intuitive view of nature and this theoretical prediction. We studied a minimal predator-prey system (a two predator-two prey system) in which the paradox of enrichment pattern can vanish; the destabilization with enrichment is reversed, leading to stabilization (a decrease in the amplitude of oscillation of population densities). For resolution of the paradox, two conditions must be met: (1) the same prey species must be preferred as a dietary item by both predator species, creating the potential for high exploitative competition between the predator species, and (2), while both predators are assumed to select their diet in accordance with optimal diet utilization theory, one predator must be a specialist and the other a generalist. In this system, the presence of a less profitable prey species can cause the increase in population oscillation amplitudes associated with increasing enrichment to be suppressed via the optimal diet utilization of the generalist predator. The resulting stabilization is explained by the mitigating effect of the less profitable prey showing better population growth with increasing enrichment on the destabilization underlying the specialist predator and prey relation, thus resolving the paradox of enrichment.     

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.