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.     

Fish as predators and prey: DNA-based assessment of their role in food webs

Fish are both consumers and prey, and as such part of a dynamic trophic network. Measuring how they are trophically linked, both directly and indirectly, to other species is vital to comprehend the mechanisms driving alterations in fish communities in space and time. Moreover, this knowledge also helps to understand how fish communities respond to environmental change and delivers important information for implementing management of fish stocks. DNA-based methods have significantly widened our ability to assess trophic interactions in both marine and freshwater systems and they possess a range of advantages over other approaches in diet analysis. In this review we provide an overview of different DNA-based methods that have been used to assess trophic interactions of fish as consumers and prey. We consider the practicalities and limitations, and emphasize critical aspects when analysing molecular derived trophic data. We exemplify how molecular techniques have been employed to unravel food web interactions involving fish as consumers and prey. In addition to the exciting opportunities DNA-based approaches offer, we identify current challenges and future prospects for assessing fish food webs where DNA-based approaches will play an important role.     

High-throughput sequencing offers insight into mechanisms of resource partitioning in cryptic bat species

Sympatric cryptic species, characterized by low morphological differentiation, pose a challenge to understanding the role of interspecific competition in structuring ecological communities. We used traditional (morphological) and novel molecular methods of diet analysis to study the diet of two cryptic bat species that are sympatric in southern England (Plecotus austriacus and P. auritus) (Fig. 1). Using Roche FLX 454 (Roche, Basel, CH) high-throughput sequencing (HTS) and uniquely tagged generic arthropod primers, we identified 142 prey Molecular Operational Taxonomic Units (MOTUs) in the diet of the cryptic bats, 60% of which were assigned to a likely species or genus. The findings from the molecular study supported the results of microscopic analyses in showing that the diets of both species were dominated by lepidopterans. However, HTS provided a sufficiently high resolution of prey identification to determine fine-scale differences in resource use. Although both bat species appeared to have a generalist diet, eared-moths from the family Noctuidae were the main prey consumed. Interspecific niche overlap was greater than expected by chance (O(jk) = 0.72, P < 0.001) due to overlap in the consumption of the more common prey species. Yet, habitat associations of nongeneralist prey species found in the diets corresponded to those of their respective bat predator (grasslands for P. austriacus, and woodland for P. auritus). Overlap in common dietary resource use combined with differential specialist prey habitat associations suggests that habitat partitioning is the primary mechanism of coexistence. The performance of HTS is discussed in relation to previous methods of molecular and morphological diet analysis. By enabling species-level identification of dietary components, the application of DNA sequencing to diet analysis allows a more comprehensive comparison of the diet of sympatric cryptic species, and therefore can be an important tool for determining fine-scale mechanisms of coexistence.     

Spatiotemporal and demographic variation in the diet of New Zealand lesser short‐tailed bats (Mystacina tuberculata)

Variation in the diet of generalist insectivores can be affected by site‐specific traits including weather, habitat, and season, as well as demographic traits such as reproductive status and age. We used molecular methods to compare diets of three distinct New Zealand populations of lesser short‐tailed bats, Mystacina tuberculata. Summer diets were compared between a southern cold‐temperate (Eglinton) and a northern population (Puroera). Winter diets were compared between Pureora and a subtropical offshore island population (Hauturu). This also permitted seasonal diet comparisons within the Pureora population. Lepidoptera and Diptera accounted for >80% of MOTUs identified from fecal matter at each site/season. The proportion of orders represented within prey and the Simpson diversity index, differed between sites and seasons within the Pureora population. For the Pureora population, the value of the Simpson diversity index was higher in summer than winter and was higher in Pureora compared to Eglinton. Summer Eglinton samples revealed that juvenile diets appeared to be more diverse than other demographic groups. Lactating females had the lowest dietary diversity during summer in Pureora. In Hauturu, we found a significant negative relationship between mean ambient temperature and prey richness. Our data suggest that M. tuberculata incorporate a narrower diversity of terrestrial insects than previously reported. This provides novel insights into foraging behavior and ecological interactions within different habitats. Our study is the first from the Southern Hemisphere to use molecular techniques to examine spatiotemporal variation in the diet of a generalist insectivore that inhabits a contiguous range with several habitat types and climates.     

Diet and foraging opportunism of the Guiana dolphin (Sotalia guianensis) in the Abrolhos Bank,Brazil

Diet analysis allows exploring how coastal dolphins interact with the environment and their role in the marine food webs. We studied the diet and feeding ecology of the Guiana dolphin, Sotalia guianensis, through analysis of stomach content from 42 animals stranded on the eastern coast of Brazil. A total of 1,336 semidigested prey items (fish, otoliths, cephalopod beaks, and crustaceans) were identified. Teleost fish comprised the most frequent food item (92% of the total), followed by cephalopods, and crustaceans. Prey belonged to 34 taxa and richness in individual stomachs varied from 1 to 15 prey taxa. Prey were generally small, but showed a significant trend to increase in size with dolphin length. The main prey of Guiana dolphins were demersal, estuarine, and sound-making fish such as catfish and sciaenids. No sex-related differences in diet were found. Diet composition varied seasonally and occurrence of prey items was coherent with breeding or high abundance periods of some fish species and squids. Our study shows the importance of demersal prey from estuarine and soft-sediment habitats to Guiana dolphin in the Abrolhos Bank and reveals that feeding habits are generalist and opportunistic, with diet reflecting the seasonal abundance and availability of prey.     

A DNA-based method for identification of krill species and its application to analysing the diet of marine vertebrate predators

Accurate identification of species that are consumed by vertebrate predators is necessary for understanding marine food webs. Morphological methods for identifying prey components after consumption often fail to make accurate identifications of invertebrates because prey morphology becomes damaged during capture, ingestion and digestion. Another disadvantage of morphological methods for prey identification is that they often involve sampling procedures that are disruptive for the predator, such as stomach flushing or lethal collection. We have developed a DNA-based method for identifying species of krill (Crustacea: Malacostraca), an enormously abundant group of invertebrates that are directly consumed by many groups of marine vertebrates. The DNA-based approach allows identification of krill species present in samples of vertebrate stomach contents, vomit, and, more importantly, faeces. Utilizing samples of faeces from vertebrate predators minimizes the impact of dietary studies on the subject animals. We demonstrate our method first on samples of Adelie penguin (Pygoscelis adeliae) stomach contents, where DNA-based species identification can be confirmed by prey morphology. We then apply the method to faeces of Adelie penguins and to faeces of the endangered pygmy blue whale (Balaenoptera musculus brevicauda). In each of these cases, krill species consumed by the predators could be identified from their DNA present in faeces or stomach contents.     

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.     

Pseudogenes and DNA-based diet analyses: a cautionary tale from a relatively well sampled predator-prey system

Mitochondrial ribosomal DNA is commonly used in DNA-based dietary analyses. In such studies, these sequences are generally assumed to be the only version present in DNA of the organism of interest. However, nuclear pseudogenes that display variable similarity to the mitochondrial versions are common in many taxa. The presence of nuclear pseudogenes that co-amplify with their mitochondrial paralogues can lead to several possible confounding interpretations when applied to estimating animal diet. Here, we investigate the occurrence of nuclear pseudogenes in fecal samples taken from bottlenose dolphins (Tursiops truncatus) that were assayed for prey DNA with a universal primer technique. We found pseudogenes in 13 of 15 samples and 1-5 pseudogene haplotypes per sample representing 5-100% of all amplicons produced. The proportion of amplicons that were pseudogenes and the diversity of prey DNA recovered per sample were highly variable and appear to be related to PCR cycling characteristics. This is a well-sampled system where we can reliably identify the putative pseudogenes and separate them from their mitochondrial paralogues using a number of recommended means. In many other cases, it would be virtually impossible to determine whether a putative prey sequence is actually a pseudogene derived from either the predator or prey DNA. The implications of this for DNA-based dietary studies, in general, are discussed.     

利用宏DNA条形码研究浮游动物多样性——以鸭绿江口为例

为评价宏DNA条形码技术在我国海洋生物多样性监测中的应用潜力,采集了22份鸭绿江口浮游动物样品,分别利用宏条形码分子鉴定和形态鉴定方法对优势类群桡足类进行多样性的比较研究。结果显示:(1)利用宏条形码分子鉴定方法共鉴定出4目23科32属229个操作分类单元(Operational Taxonomic Units, OTUs),形态方法共鉴定出3目5科5属6种;同时,利用形态鉴定得到的分类阶元多数(目:100%、科:80%、属:80%)能用宏条形码分子鉴定方法鉴定出来,而宏条形码分子鉴定方法鉴定得到的分类阶元多数(目:25%、科:83%、属:88%)却未能用形态鉴定出来,表明宏条形码分子鉴定方法在鉴定物种丰富度方面具有明显优势。(2)利用宏条形码分子鉴定与形态鉴定桡足类的多样性指数呈显著的一致性(r=0.524,P=0.024),表明宏条形码鉴定方法与形态方法在评价物种多样性方面具有较好的可比性。本研究表明宏条形码分子鉴定方法在我国海洋浮游动物业务化监测中具有较高的应用潜力。     

Foraging performance of diet-induced morphotypes in pumpkinseed sunfish (Lepomis gibbosus) favours resource polymorphism

Morphological plasticity can influence adaptive divergence when it affects fitness components such as foraging performance. We induced morphological variation in pumpkinseed sunfish (Lepomis gibbosus) ecomorphs and tested for effects on foraging performance. Young-of-year pumpkinseed sunfish from littoral and pelagic lake habitats were reared each on a 'specialist diet' representing their native habitat-specific prey, or a 'generalist diet' reflecting a combination of native and non-native prey. Specialist and generalist diets, respectively, induced divergent and intermediate body forms. Specialists had the highest capture success on their native prey whereas generalist forms were inferior. Specialists faced trade-offs across prey types. However, pelagic specialists also had the highest intake rate on both prey types suggesting that foraging trade-offs are relaxed when prey are abundant. This increases the likelihood of a resource polymorphism because the specialized pelagic form can be favoured by directional selection when prey are abundant and by diversifying selection when prey resources are restricted.     

Microbial diversity in tropical marine sediments assessed using culture-dependent and culture-independent techniques

The microbial communities associated with marine sediments are critical for ecosystem function yet remain poorly characterized. While culture-independent (CI) techniques capture the broadest perspective on community composition, culture-dependent (CD) methods can select for low abundance taxa that are missed using CI approaches. This study aimed to assess microbial diversity in tropical marine sediments at five shallow-water sites in Belize using both CD and CI techniques. The CD methods captured approximately 3% of the >800 genera detected across all sites using the CI approach. Additionally, 39 genera were only detected in culture, revealing rare taxa that were missed with the CI approach. Significantly different communities were detected across sites, with rare taxa playing an important role in distinguishing among communities. This study provides important baseline data describing shallow-water sediment microbial communities, evidence that standard cultivation techniques may be more effective than previously recognized, and the first steps towards identifying new taxa that are amenable to agar plate cultivation.     

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.     

Diet estimation based on an integrated mixed prey feeding experiment using Arctocephalus seals

Food web models depend on identifying which taxa are eaten and in what proportion they are consumed. Arctocephalus seals are generalist foragers and are an ongoing focus of Southern Hemisphere marine ecosystem research. This is the first feeding experiment to use Arctocephalus spp. to assess the utility of hard part scat analysis for diet estimation, based on mixed prey diets integrated over several days. Recovery rates of otoliths were extremely low for all taxa (0-9%). Although we could not collect scats produced during a 90 min period each day, during which the seals had access to a large pool, this result could not be attributed to otolith robustness, pinniped species or class, activity level, meal size or frequency, or fat content of the diet. We conclude that the unusually low recovery rates in this study may be due to unaccounted scats produced during 90 min of each day, if they contained otolith numbers an order of magnitude greater than all otoliths retrieved from scats produced during the other 22.5 h of each day, and/or may be related to the digestive processing of a mixed prey diet. Our study demonstrates the inadequacy of using otoliths in field collected scats for diet estimation due to the high level of unexplained variability of otolith occurrence in scats. We also identify two new potential sources of this variability. These are variability in numbers of otoliths per scat depending on activity level when a scat is excreted, and variability in recovery rates of otoliths as a function of the complexity of the diet.     

Morphological and molecular variation in tiger beetles of the Cicindela hybrida complex: is an ‘integrative taxonomy’ possible?

Current taxon assignments at the species level are frequently discordant with DNA-based analyses. Recent studies on tiger beetles in the Cicindela hybrida complex identified discordance between mtDNA patterns and the entities currently defined by the taxonomic literature. To test the accuracy of morphologically delimited groups, five named taxa (species) from 24 representative sampling sites across Europe were scored for 41 external morphological characters. Three of the named taxa were 'diagnosable', that is, defined by between one and three characters unique to each group. Newly sequenced ITS1 and existing mitochondrial cox1 markers established 20 and 22 different haplotypes, respectively, but only cox1 produced (four) diagnosable units. Phylogenetic analysis and statistical parsimony networks showed poor congruence of character variation with the taxonomic entities (and each other). Variation in morphological characters was therefore tested directly for association with DNA-based nesting groups at various hierarchical levels using permutational contingency analysis. Significant statistical associations of 11 (of 13 variable) morphological characters were observed with nesting groups from ITS1 and mitochondrial DNA markers, predominantly at the 4-step level. The analysis demonstrates the need for formal tests of congruence with morphological variation at the level of individual characters, a step that is omitted from recent studies of 'integrative taxonomy'. In addition, statistical correlation of particular morphological characters with DNA-based nesting groups can identify the lowest hierarchical level at which various character sets show congruence, as a means to define evolutionarily separated entities supported by diverse data sources.     

Morphological-dietary relationships within two assemblages of marine demersal fishes

In this study we have used morphological characters related to feeding and prey capture and dietary data to investigate the trophic organization within two assemblages of marine demersal fishes. Morphological and dietary disparities within fish assemblages were estimated from species similarities based on Euclidean distances plus species projections on the principal axes from multivariate analyses. The analyses of the morphological variables indicated that species in each assemblage comprised morphologically distinct groups strongly influenced by trophically linked characters. Stomach content analyses revealed that fish species in each assemblage were classified into three basic feeding groups: polychaete-shrimp feeders, crab feeders and fish feeders. These results indicated that food resource partitioning was operating within each assemblage. However, when morphological and trophic data were compared no significant correlations were found. The results did not particularly support the ecomorphological hypothesis that dietary differences are due to morphological differences, since similar diets do not correspond to similar morphologies. The patterns of trophic organization within the fish assemblages examined, possibly reflect differences among species due not only to the effect of ecological demands on morphology but also by their evolutionary history and constructional constraints imposed by phylogeny. This revised version was published online in August 2006 with corrections to the Cover Date.     

Diet of two large sympatric teleosts, the ling (Genypterus blacodes) and hake (Merluccius australis)

Ling and hake are tertiary consumers, and as a result both may have an important structuring role in marine communities. The diets of 2064 ling and 913 hake from Chatham Rise, New Zealand, were determined from examination of stomach contents. Ling was a benthic generalist, and hake a demersal piscivore. The diet of ling was characterised by benthic crustaceans, mainly Munida gracilis and Metanephrops challengeri, and demersal fishes, mainly Macrourids and scavenged offal from fishing vessels. The diet of hake was characterised by teleost fishes, mainly macrourids and merlucciids. Multivariate analyses using distance-based linear models found the most important predictors of diet variability were depth, fish length, and vessel type (whether the sample was collected from a commercial or research vessel) for ling, and fish length and vessel type for hake. There was no interspecific predation between ling and hake, and resource competition was largely restricted to macrourid prey, although the dominant macrourid species predated by ling and hake were different. Cluster analysis of average diet of intraspecific groups of ling and hake confirmed the persistent diet separation. Although size is a central factor in determining ecological processes, similar sized ling and hake had distinctly different foraging ecology, and therefore could influence the ecosystem in different ways, and be unequally affected by ecosystem fluctuations.     

Carnivore diet analysis based on next-generation sequencing: application to the leopard cat (Prionailurus bengalensis) in Pakistan

Diet analysis is a prerequisite to fully understand the biology of a species and the functioning of ecosystems. For carnivores, traditional diet analyses mostly rely upon the morphological identification of undigested remains in the faeces. Here, we developed a methodology for carnivore diet analyses based on the next-generation sequencing. We applied this approach to the analysis of the vertebrate component of leopard cat diet in two ecologically distinct regions in northern Pakistan. Despite being a relatively common species with a wide distribution in Asia, little is known about this elusive predator. We analysed a total of 38 leopard cat faeces. After a classical DNA extraction, the DNA extracts were amplified using primers for vertebrates targeting about 100 bp of the mitochondrial 12S rRNA gene, with and without a blocking oligonucleotide specific to the predator sequence. The amplification products were then sequenced on a next-generation sequencer. We identified a total of 18 prey taxa, including eight mammals, eight birds, one amphibian and one fish. In general, our results confirmed that the leopard cat has a very eclectic diet and feeds mainly on rodents and particularly on the Muridae family. The DNA-based approach we propose here represents a valuable complement to current conventional methods. It can be applied to other carnivore species with only a slight adjustment relating to the design of the blocking oligonucleotide. It is robust and simple to implement and allows the possibility of very large-scale analyses.     

From Puffins to Plankton: A DNA-Based Analysis of a Seabird Food Chain in the Northern Gulf of Maine

The predator-prey interactions within food chains are used to both characterize and understand ecosystems. Conventional methods of constructing food chains from visual identification of prey in predator diet can suffer from poor taxonomic resolution, misidentification, and bias against small or completely digestible prey. Next-generation sequencing (NGS) technology has become a powerful tool for diet reconstruction through barcoding of DNA in stomach content or fecal samples. Here we use multi-locus (16S and CO1) next-generation sequencing of DNA barcodes on the feces of Atlantic puffin (Fratercula arctica) chicks (n=65) and adults (n=64) and the stomach contents of their main prey, Atlantic herring (Clupea harengus, n=44) to investigate a previously studied food chain. We compared conventional and molecular-derived chick diet, tested the similarity between the diets of puffin adults and chicks, and determined whether herring prey can be detected in puffin diet samples. There was high variability in the coverage of prey groups between 16S and CO1 markers. We identified more unique prey with our 16S compared to CO1 barcoding markers (51 and 39 taxa respectively) with only 12 taxa identified by both genes. We found no significant difference between the 16S-identified diets of puffin adults (n=17) and chicks (n=41). Our molecular method is more taxonomically resolved and detected chick prey at higher frequencies than conventional field observations. Many likely planktonic prey of herring were detected in feces from puffin adults and chicks, highlighting the impact secondary consumption may have on the interpretation of molecular dietary analysis. This study represents the first simultaneous molecular investigation into the diet of multiple components of a food chain and highlights the utility of a multi-locus approach to diet reconstruction that is broadly applicable to food web analysis.     

Assessment of prey overlap between a native (Polistes humilis) and an introduced (Vespula germanica) social wasp using morphology and phylogenetic analyses of 16S rDNA

Abstract In newly invaded communities, interspecific competition is thought to play an important role in determining the success of the invader and its impact on the native community. In southern Australia, the native Polistes humilis was the predominant social wasp prior to the arrival of the exotic Vespula germanica (Hymenoptera: Vespidae). Both species forage for similar resources (water, pulp, carbohydrate and protein prey), and concerns have arisen about potential competition between them. The aim of this study was to identify the protein foods that these wasps feed on. As many prey items are masticated by these wasps to the degree that they cannot be identified using conventional means, morphological identification was complemented by sequencing fragments of the mitochondrial 16S rRNA gene. GenBank searches using blast and phylogenetic analyses were used to identify prey items to at least order level. The results were used to construct complete prey inventories for the two species. These indicate that while P. humilis is restricted to feeding on lepidopteran larvae, V. germanica collects a variety of prey of invertebrate and vertebrate origin. Calculated values of prey overlap between the two species are used to discuss the implications of V. germanica impacting on P. humilis. Results obtained are compared to those gained by solely 'conventional' methods, and the advantages of using DNA-based taxonomy in ecological studies are emphasized.