Comparing three types of dietary samples for prey DNA decay in an insect generalist predator

The rapidly growing field of molecular diet analysis is becoming increasingly popular among ecologists, especially when investigating methodologically challenging groups, such as invertebrate generalist predators. Prey DNA detection success is known to be affected by multiple factors; however, the type of dietary sample has rarely been considered. Here, we address this knowledge gap by comparing prey DNA detection success from three types of dietary samples. In a controlled feeding experiment, using the carabid beetle Pterostichus melanarius as a model predator, we collected regurgitates, faeces and whole consumers (including their gut contents) at different time points postfeeding. All dietary samples were analysed using multiplex PCR, targeting three different length DNA fragments (128, 332 and 612 bp). Our results show that both the type of dietary sample and the size of the DNA fragment contribute to a significant part of the variation found in the detectability of prey DNA. Specifically, we observed that in both regurgitates and whole consumers, prey DNA was detectable significantly longer for all fragment sizes than for faeces. Based on these observations, we conclude that prey DNA detected from regurgitates and whole consumers DNA extracts are comparable, whereas prey DNA detected from faeces, though still sufficiently reliable for ecological studies, will not be directly comparable to the former. Therefore, regurgitates and faeces constitute a useful, nonlethal source for dietary information that could be applied to field studies in situations when invertebrate predators should not be killed.     

Trophic and Non-Trophic Interactions in a Biodiversity Experiment Assessed by Next-Generation Sequencing

Plant diversity affects species richness and abundance of taxa at higher trophic levels. However, plant diversity effects on omnivores (feeding on multiple trophic levels) and their trophic and non-trophic interactions are not yet studied because appropriate methods were lacking. A promising approach is the DNA-based analysis of gut contents using next generation sequencing (NGS) technologies. Here, we integrate NGS-based analysis into the framework of a biodiversity experiment where plant taxonomic and functional diversity were manipulated to directly assess environmental interactions involving the omnivorous ground beetle Pterostichus melanarius. Beetle regurgitates were used for NGS-based analysis with universal 18S rDNA primers for eukaryotes. We detected a wide range of taxa with the NGS approach in regurgitates, including organisms representing trophic, phoretic, parasitic, and neutral interactions with P. melanarius. Our findings suggest that the frequency of (i) trophic interactions increased with plant diversity and vegetation cover; (ii) intraguild predation increased with vegetation cover, and (iii) neutral interactions with organisms such as fungi and protists increased with vegetation cover. Experimentally manipulated plant diversity likely affects multitrophic interactions involving omnivorous consumers. Our study therefore shows that trophic and non-trophic interactions can be assessed via NGS to address fundamental questions in biodiversity research.     

DNA metabarcoding multiplexing and validation of data accuracy for diet assessment: application to omnivorous diet

Ecological understanding of the role of consumer–resource interactions in natural food webs is limited by the difficulty of accurately and efficiently determining the complex variety of food types animals have eaten in the field. We developed a method based on DNA metabarcoding multiplexing and next‐generation sequencing to uncover different taxonomic groups of organisms from complex diet samples. We validated this approach on 91 faeces of a large omnivorous mammal, the brown bear, using DNA metabarcoding markers targeting the plant, vertebrate and invertebrate components of the diet. We included internal controls in the experiments and performed PCR replication for accuracy validation in postsequencing data analysis. Using our multiplexing strategy, we significantly simplified the experimental procedure and accurately and concurrently identified different prey DNA corresponding to the targeted taxonomic groups, with ≥60% of taxa of all diet components identified to genus/species level. The systematic application of internal controls and replication was a useful and simple way to evaluate the performance of our experimental procedure, standardize the selection of sequence filtering parameters for each marker data and validate the accuracy of the results. Our general approach can be adapted to the analysis of dietary samples of various predator species in different ecosystems, for a number of conservation and ecological applications entailing large‐scale population level diet assessment through cost‐effective screening of multiple DNA metabarcodes, and the detection of fine dietary variation among samples or individuals and of rare food items.     

Inferring species interactions from ecological survey data: A mechanistic approach to predict quantitative food webs of seed feeding by carabid beetles

1. Ecological networks are valuable for ecosystem analysis but their use is often limited by a lack of data because many types of ecological interaction, for example, predation, are short‐lived and difficult to observe or detect. While there are different methods for inferring the presence of interactions, they have rarely been used to predict the interaction strengths that are required to construct weighted, or quantitative, ecological networks.
2. Here, we develop a trait‐based approach suitable for inferring weighted networks, that is, with varying interaction strengths. We developed the method for seed‐feeding carabid ground beetles (Coleoptera: Carabidae) although the principles can be applied to other species and types of interaction.
3. Using existing literature data from experimental seed‐feeding trials, we predicted a per‐individual interaction cost index based on carabid and seed size. This was scaled up to the population level to create inferred weighted networks using the abundance of carabids and seeds from empirical samples and energetic intake rates of carabids from the literature. From these weighted networks, we also derived a novel measure of expected predation pressure per seed type per network.
4. This method was applied to existing ecological survey data from 255 arable fields with carabid data from pitfall traps and plant seeds from seed rain traps. Analysis of these inferred networks led to testable hypotheses about how network structure and predation pressure varied among fields.
5. Inferred networks are valuable because (a) they provide null models for the structuring of food webs to test against empirical species interaction data, for example, DNA analysis of carabid gut regurgitates and (b) they allow weighted networks to be constructed whenever we can estimate interactions between species and have ecological census data available. This permits ecological network analysis even at times and in places when interactions were not directly assessed.

     

The significance of facultative scavenging in generalist predator nutrition: detecting decayed prey in the guts of predators using PCR

Gut-content analyses using molecular techniques are an effective approach to quantifying predator-prey interactions. Predation is often assumed but scavenging is an equally likely route by which animal DNA enters the gut of a predator/scavenger. We used PCR (polymerase chain reaction) to detect scavenged material in predator gut homogenates. The rates at which DNA in decaying slugs (Mollusca: Pulmonata) and aphids (Homoptera: Aphididae) became undetectable were estimated. The detectability of DNA from both carrion types in the guts of the generalist predator Pterostichus melanarius (Coleoptera: Carabidae) was then determined. The effects of carrion age and weight, as well as beetle sex, on detection periods, were quantified. Laboratory trials measured prey preference of beetles between live and decaying prey. Further experiments measured, for the first time, feeding by P. melanarius on dead slugs and aphids directly in the field. In both field and laboratory, P. melanarius preferentially fed on dead prey if available, but preference changed as the prey became increasingly decayed. Disappearance rates for slug carrion in wheat fields and grasslands were estimated and P. melanarius was identified as the main scavenger. Comparison of the retention time for dead slugs in the field, with the detection period for decaying slug material in the guts of the predators, showed that PCR-based techniques are not able to distinguish between predated and scavenged food items. This could potentially lead to overestimation of the impact of predation on slugs (and other prey) by carabids. Possible implications of facultative scavenging by invertebrate predators for biocontrol and food-web research are discussed.     

When to use next generation sequencing or diagnostic PCR in diet analyses

Next‐generation sequencing (NGS) is increasingly used for diet analyses; however, it may not always describe diet samples well. A reason for this is that diet samples contain mixtures of food DNA in different amounts as well as consumer DNA which can reduce the food DNA characterized. Because of this, detections will depend on the relative amount and identity of each type of DNA. For such samples, diagnostic PCR will most likely give more reliable results, as detection probability is only marginally dependent on other copresent DNA. We investigated the reliability of each method to test (a) whether predatory beetle regurgitates, supposed to be low in consumer DNA, allow to retrieve prey sequences using general barcoding primers that co‐amplify the consumer DNA, and (b) to assess the sequencing depth or replication needed for NGS and diagnostic PCR to give stable results. When consumer DNA is co‐amplified, NGS is better suited to discover the range of possible prey, than for comparing co‐occurrences of diet species between samples, as retested samples were repeatedly different in prey detections with this approach. This shows that samples were incompletely described, as prey detected by diagnostic PCR frequently were missed by NGS. As the sequencing depth needed to reliably describe the diet in such samples becomes very high, the cost‐efficiency and reliability of diagnostic PCR make diagnostic PCR better suited for testing large sample‐sets. Especially if the targeted prey taxa are thought to be of ecological importance, as diagnostic PCR gave more nested and consistent results in repeated testing of the same sample.     

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.     

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.     

Revealing species-specific trophic links in soil food webs: molecular identification of scarab predators

Soil food webs are particularly important in terrestrial systems, but studying them is difficult. Here we report on the first study to apply a molecular approach to identify species-specific trophic interactions in below-ground food webs. To identify the invertebrate predator guild of the garden chafer Phyllopertha horticola (Coleoptera, Scarabaeidae) whose root-feeding larvae can be highly abundant in grasslands, a specific DNA marker was developed. It allowed detection of P. horticola egg and white grub meals within the gut content of Poecilus versicolor (Coleoptera, Carabidae) larvae for up to 24 h post-feeding. Soil samples from an alpine grassland revealed a diverse below-ground macro-invertebrate community with earthworms, P. horticola larvae, and centipedes as well as beetle larvae as the most abundant detritivores, herbivores, and predators, respectively. Garden chafer DNA was detected in 18.6%, 4.1%, and 4.4% of field-collected Geophilidae (n = 124), beetle larvae (n = 159), and Lithobiidae (n = 49), respectively. We conclude that most of the investigated predators actively preyed on P. horticola, as secondary predation is unlikely to be detected in below-ground systems. Moreover, scavenging most likely contributes only to a small percentage of the revealed trophic links due to the low availability of carrion. Sampling date did not influence prey detection rates, indicating that both P. horticola eggs and larvae were preyed on. Only 2.7% of the below-ground predators tested positive for earthworms, an alternative, highly abundant prey, suggesting that P. horticola represents an important prey source for centipedes and predatory beetle larvae during summer within the soil food web.     

Factors affecting detectability of prey DNA in the gut contents of invertebrate predators: a polymerase chain reaction-based method

This study aimed to determine factors that influence the detection of DNA of Plutella xylostella L. (Lepidoptera: Plutellidae) in the gut contents of arthropod predators when the polymerase chain reaction is used to amplify a diagnostic fragment of the gene coding for cytochrome oxidase subunit I. The effects of temperature, time since feeding, subsequent food intake, sex, weight, and species of predator on prey detectability were studied in the laboratory. Three types of predator were studied: the spider Venator spenceri Hogg. (Araneae: Lycosidae), a bug with sucking mouthparts, Nabis kinbergii (Reuter) (Heteroptera: Nabidae), and a coccinellid with chewing mouthparts, Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae). In all experiments, the detectability of prey DNA was negatively correlated with time post‐feeding. The duration of detectability differed among the predator species. The time calculated for median detection success at 20 °C ranged from 49.6 h in V. spenceri to 36.1 h in N. kinbergii and 17.1 h in H. variegata. In H. variegata, but not in V. spenceri, the rate of detection decreased with increasing temperature. Subsequent food intake did not affect the detectability of DNA of P. xylostella in V. spenceri. In H. variegata, sex and weight of the predator did not influence detection of prey DNA. In addition, this study uncovered potential sources of error caused by detection of prey DNA following secondary cannibalistic and intraguild predation. The results provide essential information for the interpretation of prey detection data from field‐collected predators’ gut contents.     

Development of specific ITS markers for plant DNA identification within herbivorous insects

DNA-based techniques have proved to be very useful methods to study trophic relationships between pests and their natural enemies. However, most predators are best defined as omnivores, and the identification of plant-specific DNA should also allow the identification of the plant species the predators have been feeding on. In this study, a PCR approach based on the development of specific primers was developed as a self-marking technique to detect plant DNA within the gut of one heteropteran omnivorous predator (Macrolophus pygmaeus) and two lepidopteran pest species (Helicoverpa armigera and Tuta absoluta). Specific tomato primers were designed from the ITS 1-2 region, which allowed the amplification of a tomato DNA fragment of 332?bp within the three insect species tested in all cases (100% of detection at t=0) and did not detect DNA of other plants nor of the starved insects. Plant DNA half-lives at 25°C ranged from 5.8?h, to 27.7?h and 28.7?h within M. pygmaeus, H. armigera and T. absoluta, respectively. Tomato DNA detection within field-collected M. pygmaeus suggests dietary mixing in this omnivorous predator and showed a higher detection of tomato DNA in females and nymphs than males. This study provides a useful tool to detect and to identify plant food sources of arthropods and to evaluate crop colonization from surrounding vegetation in conservation biological control programs.     

Free‐living nematodes as prey for higher trophic levels of forest soil food webs

Nematodes are the most abundant invertebrates in soils and are key prey in soil food webs. Uncovering their contribution to predator nutrition is essential for understanding the structure of soil food webs and the way energy channels through soil systems. Molecular gut content analysis of consumers of nematodes, such as soil microarthropods, using specific DNA markers is a novel approach for studying predator–prey interactions in soil. We designed new specific primer pairs (partial 18S rDNA) for individual soil‐living bacterial‐feeding nematode taxa (Acrobeloides buetschlii, Panagrellus redivivus, Plectus velox and Plectus minimus). Primer specificity was tested against more than 100 non‐target soil organisms. Further, we determined how long nematode DNA can be traced in the gut of predators. Potential predators were identified in laboratory experiments including nine soil mite (Oribatida, Gamasina and Uropodina) and ten springtail species (Collembola). Finally, the approach was tested under field conditions by analyzing five mite and three collembola species for feeding on the three target nematode species. The results proved the three primer sets to specifically amplify DNA of the respective nematode taxa. Detection time of nematode DNA in predators varied with time of prey exposure. Further, consumption of nematodes in the laboratory varied with microarthropod species. Our field study is the first definitive proof that free‐living nematodes are important prey for a wide range of soil microarthropods including those commonly regarded as detritivores. Overall, the results highlight the eminent role of nematodes as prey in soil food webs and for channelling bacterial carbon to higher trophic levels.     

Beyond barcodes: complex DNA taxonomy of a South Pacific Island radiation

DNA barcodes can provide rapid species identification and aid species inventories in taxonomically unstudied groups. However, the approach may fail in recently diverged groups with complex gene histories, such as those typically found on oceanic islands. We produced a DNA-based inventory of taxonomically little known diving beetles (genus Copelatus) in the Fiji archipelago, where they are a dominant component of the aquatic invertebrate fauna. Sampling from 25 localities on five islands and analysis of sequences from one nuclear (328bp histone 3) and three mitochondrial (492bp rrnL, 786bp cox1, 333bp cob) gene regions revealed high haplotype diversity, mainly originated since the Pleistocene, and subdivided into three major phylogenetic lineages and 22 statistical parsimony networks. A traditional taxonomic study delineated 25 morphologically defined species that were largely incongruent with the DNA-based groups. Haplotype diversity and their spatial arrangement demonstrated a continuum of relatedness in Fijian Copelatus, with evidence for introgression at various hierarchical levels. The study illustrates the difficulties for formal classification in evolutionarily complex lineages, and the potentially misleading conclusions obtained from either DNA barcodes or morphological traits alone. However, the sequence profile of Fijian Copelatus provides an evolutionary framework for the group and a DNA-based reference system for the integration of ecological and other biodiversity data, independent of the Linnaean naming system.     

Nest and foraging‐site selection in Yellowhammers Emberiza citrinella: implications for chick provisioning

Capsule Vegetation structure and invertebrate abundance interact to influence both foraging sites and nestling provisioning rate; when invertebrate availability is low, adults may take greater risks to provide food for their young.

Aims To investigate nesting and foraging ecology in a declining farmland bird whose fledging success is influenced by the availability of invertebrate prey suitable for feeding to offspring, and where perceived predation risk during foraging can be mediated by vegetation structure.

Methods Provisioning rates of adult Yellowhammers feeding nestlings were measured at nests on arable farmland. Foraging sites were compared with control sites of both the same and different microhabitats; provisioning rate was related to habitat features of foraging‐sites.

Results Foraging sites had low vegetation density, probably enhancing detection of predators, or high invertebrate abundance at high vegetation density. Parental provisioning rate decreased with increasing vegetation cover at foraging sites with high invertebrate abundance; conversely, where invertebrate abundance was low, provisioning rate increased with increasing vegetation cover.

Conclusions Vegetation structure at foraging sites suggests that a trade‐off between predator detection and prey availability influences foraging site selection in Yellowhammers. Associations between parental provisioning rate and vegetation variables suggest that where invertebrate abundance is high birds increase time spent scanning for predators at higher vegetation densities; however, when prey are scarce, adults may take more risks to provide food for their young.     

Effects of consumer surface sterilization on diet DNA metabarcoding data of terrestrial invertebrates in natural environments and feeding trials

1. DNA metabarcoding is an emerging tool used to quantify diet in environments and consumer groups where traditional approaches are unviable, including small‐bodied invertebrate taxa. However, metabarcoding of small taxa often requires DNA extraction from full body parts (without dissection), and it is unclear whether surface contamination from body parts alters presumed diet presence or diversity.
2. We examined four different measures of diet (presence, rarefied read abundance, richness, and species composition) for a terrestrial invertebrate consumer (the spider Heteropoda venatoria) both collected in its natural environment and fed an offered diet item in contained feeding trials using DNA metabarcoding of full body parts (opisthosomas). We compared diet from consumer individuals surface sterilized to remove contaminants in 10% commercial bleach solution followed by deionized water with a set of unsterilized individuals.
3. We found that surface sterilization did not significantly alter any measure of diet for consumers in either a natural environment or feeding trials. The best‐fitting model predicting diet detection in feeding trial consumers included surface sterilization, but this term was not statistically significant (β = −2.3, p‐value = .07).
4. Our results suggest that surface contamination does not seem to be a significant concern in this DNA diet metabarcoding study for consumers in either a natural terrestrial environment or feeding trials. As the field of diet DNA metabarcoding continues to progress into new environmental contexts with various molecular approaches, we suggest ongoing context‐specific consideration of the possibility of surface contamination.

     

The effects of temperature on detection of prey DNA in two species of carabid beetle

PCR-based techniques to investigate predator-prey trophic interactions are starting to be used more widely, but factors affecting DNA decay in predator guts are still poorly understood. Here, we investigated the effects of time since feeding, temperature and amplicon size on the detectability of prey DNA in the gut content of two closely related predator species. Cereal aphids, Sitobion avenae, were fed to the carabid beetles Pterostichus melanarius and Nebria brevicollis. Beetles were allowed to digest their meal at 12 degrees C, 16 degrees C and 20 degrees C, and batches of beetles were subsequently frozen at time periods from 0-72 h after feeding. Aphid DNA was detected within beetles' gut contents using primers amplifying fragments of 85, 231, 317 and 383 bp. Prey DNA detection rates were significantly higher in N. brevicollis than in P. melanarius, indicating fundamental dissimilarities in prey digestion capacities. High temperatures (20 degrees C) and large amplicons (383 bp) significantly decreased detection rates. The shortest amplicon gave the highest prey DNA detection success, whereas no differences were observed between the 231 bp and the 317 bp fragment. Our results indicate that factors such as ambient temperature, predator taxon and amplicon size should all be considered when interpreting data derived from PCR-based prey detection. Correction for such factors should make calculation of predation rates in the field more accurate and could help us to estimate when predation events occur in the field.     

Molecular diet analysis in mussels and other metazoan filter feeders and an assessment of their utility as natural eDNA samplers

Molecular gut content analysis is a popular tool to study food web interactions and has recently been suggested as an alternative source for DNA-based biomonitoring. However, the overabundant consumer's DNA often outcompetes that of its diet during PCR. Lineage-specific primers are an efficient means to reduce consumer amplification while retaining broad specificity for dietary taxa. Here, we designed an amplicon sequencing assay to monitor the eukaryotic diet of mussels and other metazoan filter feeders and explore the utility of mussels as natural eDNA samplers to monitor planktonic communities. We designed several lineage-specific rDNA primers with broad taxonomic suitability for eukaryotes. The primers were tested using DNA extracts of different limnic and marine mussel species and the results compared to eDNA water samples collected next to the mussel colonies. In addition, we analysed several 25-year time series samples of mussels from German rivers. Our primer sets efficiently prevent the amplification of mussels and other metazoans. The recovered DNA reflects a broad dietary preference across the eukaryotic tree of life and considerable taxonomic overlap with filtered water samples. We also show the utility of a reversed version of our primers, which prevents amplification of nonmetazoan taxa from complex eukaryote community samples, by enriching fauna associated with the marine brown algae Fucus vesiculosus. Our protocol will enable large-scale dietary analysis in metazoan filter feeders, facilitate aquatic food web analysis and allow surveying of aquacultures for pathogens. Moreover, we show that mussels and other aquatic filter feeders can serve as complementary DNA source for biomonitoring.     

‘Direct PCR’ optimization yields a rapid,cost‐effective,nondestructive and efficient method for obtaining DNA barcodes without DNA extraction

Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction (‘direct PCR’) and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands.     

Predicting and testing resource partitioning in a tropical fish assemblage of zooplanktivorous 'barbs': an ecomorphological approach

Morphometrics on 25 critical feeding structures predicted conspicuous specializations in Barbus tanapelagius (pursuit hunting for zooplankton), Labeobarbus brevicephalus (surface dwelling pump-filter-feeder on zooplankton) and Barbus pleurogramma (particulate feeding on tough, benthic food), whereas for Barbus humilis intermediate values predicted few constraints and specializations in feeding. These potential niches, set by fish size and structural constraints, were tested by comparing gut contents collected during a 24 months sampling programme on Lake Tana, Ethiopia. Zooplankton dominated the diet of B. tanapelagius (75% of gut volume) and L. brevicephalus (39%). The guts of B. pleurogramma and juveniles of the large labeobarbs showed an array of benthic food types, whereas B. humilis had the widest food niche, both zooplankton (40% of gut volume) and benthic invertebrates. Although the pelagic species showed the largest spatial overlap, their size, feeding modes and utilization of zooplankters differed: L. brevicephalus preyed predominantly on the larger zooplankton ( Daphnia sp.) and B. tanapelagius also on smaller species ( e.g. Bosmina sp. and cyclopoid copepods). The spatial segregation between B. tanapelagius (pelagic) and the juvenile labeobarbs (littoral) indicated the possibility for a small pelagic barb fishery without negative effects on the labeobarb stocks. The ecomorphological approach using the 'Food-Fish Model' appeared to predict competitive positions and resource partitioning appropriately, and is of major importance to evaluate food web interactions.     

Infiltration by alien predators into invertebrate food webs in Hawaii: a molecular approach

Abstract Alien invertebrate predators have been introduced to Hawaii to control pests, particularly in lowland areas where most crops are grown. We developed techniques for assessing the impact of these predators on native food webs in relatively pristine upland areas where, it was hypothesized, few lowland predators might be found. Predator densities were assessed along transects within the Alakaii Swamp on Kaua'i. The most numerous alien biocontrol agents found were Halmus chalybeus (Coccinellidae), a species known to feed on Lepidoptera eggs. Laboratory experiments were conducted using two genera of endemic Lepidoptera, Scotorythra and Eupithecia (Geometridae), that are of considerable conservation value, the former because of its recent speciation across Hawaii, the latter for its unique predatory larvae. Techniques were developed for detecting Lepidoptera DNA within the guts of alien predators using prey-specific PCR primers. General primers amplified fragments of the mitochondrial cytochrome oxidase I gene from beetles and Lepidoptera. The sequences were aligned and used successfully to design target-specific primers for general detection of the remains of Geometridae and for particular species, including Scotorythra rara and Eupithecia monticolans. DNA fragments amplified were short [140-170 base pairs (bp)], optimizing detection periods following prey ingestion. Trials using the introduced biocontrol agent Curinus coeruleus (Coccinellidae) demonstrated detection of Lepidoptera DNA fragments = 151 bp in 85-100% of beetles after 24 h digestion of an early instar larva. This study provides a framework for future use of molecular gut analysis in arthropod conservation ecology and food web research with considerable potential for quantifying threats to endemic species in Hawaii and elsewhere.