Friday, March 16, 2018

Weekend reads

More to read for you in case you follow my recommendations. As stated before I am posting only a selection and all papers are chosen at least in part based on subjective criteria. So, here we go, my take on what I think you (and especially my students) should read ;-)

The biocide Bacillus thuringiensis var. israelensis (Bti) is widely applied for mosquito control in temporary wetlands of the German Upper Rhine Valley. Even though Bti is considered environmentally friendly, several studies have shown non-target effects on chironomids, a key food resource in wetland ecosystems. Chironomids have been proposed as important indicators for monitoring freshwater ecosystems, however, morphological determination is very challenging. In this study, we investigated the effectiveness of metabarcoding for chironomid diversity assessment and tested the retrieved chironomid operational taxonomic units (OTUs) for possible changes in relative abundance and species diversity in relation to mosquito control actions in four temporary wetlands. Three of these wetlands were, for the first year after 20 years of Bti treatment, partly left Bti-untreated in a split field design, and one wetland has never been treated with Bti. Our metabarcoding approach detected 54 chironomid OTUs across all study sites, of which almost 70% could be identified to species level comparisons against the BOLD database. We showed that metabarcoding increased chironomid species determination by 70%. However, we found only minor significant effects of Bti on the chironomid community composition, even though Bti reduced chironomid emergence by 65%. This could be due to a time lag of chironomid recolonization, since the study year was the first year of Bti intermittence after about 20 years of Bti application in the study area. Subsequent studies will have to address if and how the chironomid community composition will recover further in the now Bti-untreated temporary wetlands to assess effects of Bti.

The introduction of domesticated animals into new environments can lead to considerable ecological disruption, and it can be difficult to predict their impact on the new ecosystem. In this study, we use faecal metabarcoding to characterize the diets of three ruminant taxa in the rangelands of south-western New South Wales, Australia. Our study organisms included goats (Capra aegagrus hircus) and two breeds of sheep (Ovis aries): Merinos, which have been present in Australia for over two hundred years, and Dorpers, which were introduced in the 1990s. We used High-Throughput Sequencing methods to sequence the rbcL and ITS2 genes of plants in the faecal samples, and identified the samples using the GenBank and BOLD online databases, as well as a reference collection of sequences from plants collected in the study area. We found that the diets of all three taxa were dominated by the family Malvaceae, and that the Dorper diet was more diverse than the Merino diet at both the family and the species level. We conclude that Dorpers, like Merinos, are potentially a threat to some vulnerable species in the rangelands of New South Wales.

Effective ecosystem conservation and resource management require quantitative monitoring of biodiversity, including accurate descriptions of species composition and temporal variations of species abundance. Accordingly, quantitative monitoring of biodiversity has been performed for many ecosystems, but it is often time- and effort-consuming and costly. Recent studies have shown that environmental DNA (eDNA), which is released to the environment from macro-organisms living in a habitat, contains information about species identity and abundance. Thus, analysing eDNA would be a promising approach for more efficient biodiversity monitoring. In the present study, internal standard DNAs (i.e. known amounts of short DNA fragments from fish species that have never been observed in a sampling area) were added to eDNA samples, which were collected weekly from a coastal marine ecosystem in Maizuru Bay, Japan (from April 2015 to March 2016) and metabarcoding analysis was performed using Illumina MiSeq to simultaneously identify fish species and quantify fish eDNA copy numbers. A correction equation was obtained for each sample using the relationship between the number of sequence reads and the added amount of the standard DNAs and this equation was used to estimate the copy numbers from the sequence reads of non-standard fish eDNA. The calculated copy numbers showed significant positive correlations with those determined by quantitative PCR, suggesting that eDNA metabarcoding with standard DNA enabled useful quantification of eDNA. Furthermore, for samples that show a high level of PCR inhibition, this method might allow more accurate quantification than qPCR because the correction equations generated using internal standard DNAs would include the effect of PCR inhibition. A single run of Illumina MiSeq produced >70 quantitative fish eDNA time series in this study, showing that this method could contribute to more efficient and quantitative monitoring of biodiversity.

Freshwater metazoan biodiversity assessment using environmental DNA (eDNA) captured on filters offers new opportunities for water quality management. Filtering of water in the field is a logistical advantage compared to transport of water to the nearest lab, and thus, appropriate filter preservation becomes crucial for maximum DNA recovery. Here, the effect of four different filter preservation strategies, two filter types, and pre-filtration were evaluated by measuring metazoan diversity and community composition, using eDNA collected from a river and a lake ecosystem. The filters were preserved cold on ice, in ethanol, in lysis buffer and dry in silica gel. Our results show that filters preserved either dry or in lysis buffer give the most consistent community composition. In addition, mixed cellulose ester filters yield more consistent community composition than polyethersulfone filters, while the effect of pre-filtration remained ambiguous. Our study facilitates development of guidelines for aquatic community-level eDNA biomonitoring, and we advocate filtering in the field, using mixed cellulose ester filters and preserving the filters either dry or in lysis buffer.

We introduce a method for assigning names to CO1 metabarcode sequences with confidence scores in a rapid, high-throughput manner. We compiled nearly 1 million CO1 barcode sequences appropriate for classifying arthropods and chordates. Compared to our previous Insecta classifier, the current classifier has more than three times the taxonomic coverage, including outgroups, and is based on almost five times as many reference sequences. Unlike other popular rDNA metabarcoding markers, we show that classification performance is similar across the length of the CO1 barcoding region. We show that the RDP classifier can make taxonomic assignments about 19 times faster than the popular top BLAST hit method and reduce the false positive rate from nearly 100% to 34%. This is especially important in large-scale biodiversity and biomonitoring studies where datasets can become very large and the taxonomic assignment problem is not trivial. We also show that reference databases are becoming more representative of current species diversity but that gaps still exist. We suggest that it would benefit the field as a whole if all investigators involved in metabarocoding studies, through collaborations with taxonomic experts, also planned to barcode representatives of their local biota as a part of their projects.

Birds play unique functional roles in the maintenance of ecosystems, such as pollination and seed dispersal, and thus monitoring bird species diversity is a first step towards avoiding undesirable consequences of anthropogenic impacts on bird communities. In the present study, we hypothesized that birds, regardless of their main habitats, must have frequent contact with water and that tissues that contain their DNA that persists in the environment (environmental DNA; eDNA) could be used to detect the presence of avian species. To this end, we applied a set of universal PCR primers (MiBird, a modified version of fish/mammal universal primers) for metabarcoding avian eDNA. We confirmed the versatility of MiBird primers by performing in silico analyses and by amplifying DNAs extracted from bird tissues. Analyses of water samples from zoo cages of birds with known species composition suggested that the use of MiBird primers combined with Illumina MiSeq could successfully detect avian species from water samples. Additionally, analysis of water samples collected from a natural pond detected five avian species common to the sampling areas. The present findings suggest that avian eDNA metabarcoding would be a complementary detection/identification tool in cases where visual census of bird species is difficult.

Anthropogenic plastic pollution is a global problem. In the marine environment, one of its less studied effects is the transport of attached biota, which might lead to introductions of non-native species in new areas or aid in habitat expansions of invasive species. The goal of the present work was to assess if the material composition of beached anthropogenic litter is indicative of the rafting fauna in a coastal area and could thus be used as a simple and cost-efficient tool for risk assessment in the future. Beached anthropogenic litter and attached biota along the 200 km coastline of Asturias, central Bay of Biscay, Spain, were analysed. The macrobiotic community attached to fouled litter items was identified using genetic barcoding combined with visual taxonomic analysis, and compared between hard plastics, foams, other plastics and non-plastic items. On the other hand, the material composition of beached litter was analysed in a standardized area on each beach. From these two datasets, the expected frequency of several rafting taxa was calculated for the coastal area and compared to the actually observed frequencies. The results showed that plastics were the most abundant type of beached litter. Litter accumulation was likely driven by coastal sources (industry, ports) and river/sewage inputs and transported by near-shore currents. Rafting vectors were almost exclusively made up of plastics and could mainly be attributed to fishing activity and leisure/ household. We identified a variety of rafting biota, including species of goose barnacles, acorn barnacles, bivalves, gastropods, polychaetes and bryozoan, and hydrozoan colonies attached to stranded litter. Several of these species were non-native and invasive, such as the giant Pacific oyster (Crassostrea gigas) and the Australian barnacle (Austrominius modestus). The composition of attached fauna varied strongly between litter items of different materials. Plastics, except for foam, had a much more diverse attached community than non-plastic materials. The predicted frequency of several taxa attached to beached litter significantly correlated with the actually observed frequencies. Therefore we suggest that the composition of stranded litter on a beach or an area could allow for predictions about the corresponding attached biotic community, including invasive species.

Thursday, March 15, 2018

Saving endangered species with an app

An app developed with support from USAID is making wildlife protection officers more effective in their efforts to combat wildlife trafficking in Southeast Asia. WildScan, a mobile species identification and response app, is designed to help law enforcement officials respond to wildlife trafficking, an illicit trade estimated at $19 billion per year and run by organized criminal syndicates. WildScan is currently available for free on Android devices and available in English, Thai and Vietnamese. The app will soon be available in Bahasa Indonesia, Bahasa Malaysia and Khmer in addition to updated Android and new Apple iOS versions.

The app is a result of a collaborative partnership between academics, law enforcement, scientists and other wildlife specialists. USAID worked through its implementing partner, Freeland, under the USAID Asia’s Regional Response to Endangered Species Trafficking program.

Law enforcement agencies, as well as casual phone users, can now identify endangered species using high-resolution photos and the app allows them to report illegal collections of terrestrial, marine, and freshwater animals to the proper authorities. Wildscan not only includes clues to identify over 700 protected species but also offers primary care tips for injured animals.

Here is a video showcasing the app.

Thursday, March 8, 2018

Weekend reads

Another week, another pile of reading material. This time with some bioinformatics. Enjoy!

In recent years, molecular species delimitation has become a routine approach for quantifying and classifying biodiversity. Barcoding methods are of particular importance in large-scale surveys as they promote fast species discovery and biodiversity estimates. Among those, distance-based methods are the most common choice as they scale well with large datasets; however, they are sensitive to similarity threshold parameters and they ignore evolutionary relationships. The recently introduced "Poisson Tree Processes" (PTP) method is a phylogeny-aware approach that does not rely on such thresholds. Yet, two weaknesses of PTP impact its accuracy and practicality when applied to large datasets; it does not account for divergent intraspecific variation and is slow for a large number of sequences.
We introduce the multi-rate PTP (mPTP), an improved method that alleviates the theoretical and technical shortcomings of PTP. It incorporates different levels of intraspecific genetic diversity deriving from differences in either the evolutionary history or sampling of each species. Results on empirical data suggest that mPTP is superior to PTP and popular distance-based methods as it, consistently yields more accurate delimitations with respect to the taxonomy (i.e., identifies more taxonomic species, infers species numbers closer to the taxonomy). Moreover, mPTP does not require any similarity threshold as input. The novel dynamic programming algorithm attains a speedup of at least five orders of magnitude compared to PTP, allowing it to delimit species in large (meta-) barcoding data. In addition, Markov Chain Monte Carlo sampling provides a comprehensive evaluation of the inferred delimitation in just a few seconds for millions of steps, independently of tree size.
mPTP is implemented in C and is available for download at under the GNU Affero 3 license. A web-service is available at

The body of DNA sequence data lacking taxonomically informative sequence headers is rapidly growing in user and public databases (e.g. sequences lacking identification and contaminants). In the context of systematics studies, sorting such sequence data for taxonomic curation and/or molecular diversity characterization (e.g. crypticism) often requires the building of exploratory phylogenetic trees with reference taxa. The subsequent step of segregating DNA sequences of interest based on observed topological relationships can represent a challenging task, especially for large datasets.
We have written TREE2FASTA, a Perl script that enables and expedites the sorting of FASTA-formatted sequence data from exploratory phylogenetic trees. TREE2FASTA takes advantage of the interactive, rapid point-and-click color selection and/or annotations of tree leaves in the popular Java tree-viewer FigTree to segregate groups of FASTA sequences of interest to separate files. TREE2FASTA allows for both simple and nested segregation designs to facilitate the simultaneous preparation of multiple data sets that may overlap in sequence content.

Biological soil crusts (BSCs) are amalgamations of autotrophic, heterotrophic and saprotrophic organisms. In the Polar Regions, these unique communities occupy essential ecological functions such as primary production, nitrogen fixation and ecosystem engineering. Here we present the first molecular survey of BSCs from the Arctic and Antarctica focused on both eukaryotes and prokaryotes as well as passive and active biodiversity. Considering sequence abundance, Bryophyta is among the most abundant taxa in all analyzed BSCs suggesting that they were in a late successional stage. In terms of algal and cyanobacterial biodiversity, the genera Chloromonas, Coccomyxa, Elliptochloris and Nostoc were identified in all samples regardless of origin confirming their ubiquitous distribution. For the first time, we found the chrysophyte Spumella to be common in polar BSCs as it was present in all analyzed samples. Co-occurrence analysis revealed the presence of sulfur metabolizing microbes indicating that BSCs also play an important role for the sulfur cycle. In general, phototrophs were most abundant within the BSCs but there was also a diverse community of heterotrophs and saprotrophs. Our results show that BSCs are unique microecosystems in polar environments with an unexpectedly high biodiversity.

eDNA metabarcoding represents a new tool for community biodiversity assessment in a broad range of aquatic and terrestrial habitats. However, much of the existing literature focuses on methodological development rather than testing of ecological hypotheses. Here, we use presence-absence data generated by eDNA metabarcoding of over 500 UK ponds to examine: 1) species associations between the great crested newt (Triturus cristatus) and other vertebrates, 2) determinants of great crested newt occurrence at the pondscape, and 3) determinants of vertebrate species richness at the pondscape. The great crested newt was significantly associated with nine vertebrate species. Occurrence in ponds was broadly reduced by more fish species, but enhanced by more waterfowl and other amphibian species. Abiotic determinants (including pond area, depth, and terrestrial habitat) were identified, which both corroborate and contradict existing literature on great crested newt ecology. Some of these abiotic factors (pond outflow) also determined species richness at the pondscape, but other factors were unique to great crested newt (pond area, depth, and ruderal habitat) or the wider biological community (pond density, macrophyte cover, terrestrial overhang, rough grass habitat, and overall terrestrial habitat quality) respectively. The great crested newt Habitat Suitability Index positively correlated with both eDNA-based great crested newt occupancy and vertebrate species richness. Our study is one of the first to use eDNA metabarcoding to test abiotic and biotic determinants of pond biodiversity. eDNA metabarcoding provided new insights at scales that were previously unattainable using established methods. This tool holds enormous potential for testing ecological hypotheses alongside biodiversity monitoring and pondscape management.

A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world's oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals.

Tuesday, March 6, 2018

Get them before they get you

Haemadipsa sp, Credit: AMNH/M. Siddall
Southern Asia is a biodiversity hotspot both for terrestrial mammals and for leeches. Many small-mammal groups are under-studied in this region, while other mammals are of known conservation concern. In addition to standard methods for surveying mammals, it has recently been demonstrated that residual bloodmeals within leeches can be sequenced to find mammals in a given area. While these invertebrate-parasite-derived DNA (iDNA) methods are promising, most of the leech species utilized for this type of survey remain unevaluated, notwithstanding that their diversity varies substantially.

Researchers at the American Museum of Natural History, conducted a broad survey across southern Asia to reinforce the idea that the mammal biodiversity of an area can be determined by looking at the DNA found in leeches' blood meals. 

The usefulness of iDNA was first shown in an earlier study on about two dozen leeches found in Vietnam. This new survey collected and genetically analyzed about 750 terrestrial leeches in the genus Haemadipsa from the forests of Bangladesh, Cambodia, and China. The colleagues found that the leeches feed at least somewhat indiscriminately on a large variety of mammals, including small deer called muntjacs, macaque monkeys, wildcats, rodents like porcupines and rats, as well as a vulnerable species in the area, a gaur, or Indian bison. They also recovered DNA from three types of ground-dwelling birds and one species of bat. So far there were only a few previous and somewhat anecdotal reports of those animals being targeted by Haemadipsa leeches.

This work is turning out to be an extremely useful tool for conservation purposes, and it's quick and easy to survey a park in this way as you don't really need to search for the leeches-they come to you looking for a meal. You just go on a casual hike and make sure you get the leeches before they get you. A snapshot of the vertebrates in an area can be taken with just one day's worth of sampling; the current standard for surveys, camera traps, takes months or longer.

Friday, March 2, 2018

Weekend reads

Another Friday, another set of papers to enjoy over the weekend. All related to DNA barcoding, one way or another. I should also stress that my weekly selection of articles is of course rather subjective. I pick what I consider interesting and sometimes my choices are not necessarily based on the fact that I share opinions or interpretations. On the contrary, you occasionally find papers here that I find frustrating and going in the wrong direction, e.g. the first one last week which is a good example of a paper where I find both question, results and general approach good and very interesting but when it comes to the choice of methods I am at a loss. This study could have been done with standard COI barcodes if only somebody had bothered to put some effort into primer development.

Aphids are viral vectors in potatoes, most importantly of Potato virus Y (PVY), and insecticides are frequently used to reduce viral spread during the crop season. Aphids collected from the potato belt of New Brunswick, Canada, in 2015 and 2016 were surveyed for known and novel mutations in the Na-channel (para) gene, coding for the target of synthetic pyrethroid insecticides. Specific genetic mutations known to confer resistance (kdr and skdr) were found in great abundance in Myzus persicae (Sulzer) (Hemiptera: Aphididae), which rose from 76% in 2015 to 96% in 2016. Aphids other than M. persicae showed lower frequency of resistance. In 2015, 3% of individuals contained the resistance mutation skdr, rising to 13% in 2016 (of 45 species). Several novel resistance mutations or mutations not before reported in aphids were identified in this gene target. One of these mutations, I936V, is known to confer pyrethroid resistance in another unrelated insect, and three others occur immediately adjacent and prompt similar chemical shifts in the primary protein structure, to previously characterized mutations associated with pyrethroid resistance. Most novel mutations were found in species other than M. persicae or others currently tracked individually by the provincial aphid monitoring program, which were determined by cytochrome C oxidase I (cox1) sequencing. Through our cox1 DNA barcoding survey, at least 45 species of aphids were discovered in NB potato fields in 2015 and 2016, many of which are known carriers of PVY.

Determining the host-parasitoid interactions and parasitism rates for invasive species entering novel environments is an important first step in assessing potential routes for biocontrol and integrated pest management. Conventional insect rearing techniques followed by taxonomic identification are widely used to obtain such data, but this can be time consuming and prone to biases. Here we present a Next Generation Sequencing approach for use in ecological studies which allows for individual level metadata tracking of large numbers of invertebrate samples through the use of hierarchically organised molecular identification tags. We demonstrate its utility using a sample data set examining both species identity and levels of parasitism in late larval stages of the Oak Processionary Moth (Thaumetopoea processionea - Linn. 1758), an invasive species recently established in the UK. Overall we find that there are two main species exploiting the late larval stages of Oak Processionary Moth in the UK with the main parasitoid (Carcelia iliaca - Ratzeburg, 1840) parasitising 45.7% of caterpillars, while a rare secondary parasitoid (Compsilura conccinata - Meigen, 1824) was also detected in 0.4% of caterpillars. Using this approach on all life stages of the Oak Processionary Moth may demonstrate additional parasitoid diversity. We discuss the wider potential of nested tagging DNA-metabarcoding for constructing large, highly-resolved species interaction networks. 

A cross-taxa study using environmental DNA/RNA metabarcoding to measure biological impacts of offshore oil and gas drilling and production operations.
Standardized ecosystem-based monitoring surveys are critical for providing information on marine ecosystem health. Environmental DNA/RNA (eDNA/eRNA) metabarcoding may facilitate such surveys by quickly and effectively characterizing multi-trophic levels. In this study, we assessed the suitability of eDNA/eRNA metabarcoding to evaluate changes in benthic assemblages of bacteria, Foraminifera and other eukaryotes along transects at three offshore oil and gas (O&G) drilling and production sites, and compared these to morphologically characterized macro-faunal assemblages. Bacterial communities were the most responsive to O&G activities, followed by Foraminifera, and macro-fauna (the latter assessed by morphology). The molecular approach enabled detection of hydrocarbon degrading taxa such as the bacteria Alcanivorax and Microbulbifer at petroleum impacted stations. Most identified indicator taxa, notably among macro-fauna, were highly specific to site conditions. Based on our results we suggest that eDNA/eRNA metabarcoding can be used as a stand-alone method for biodiversity assessment or as a complement to morphology-based monitoring approaches.

We analysed with multigene (18S and COI) metabarcoding the effects of the proliferation of invasive seaweeds on rocky littoral communities in two Spanish Marine Protected Areas. The invasive algae studied were Caulerpa cylindracea, Lophocladia lallemandii and Asparagopsis armata. They are canopy-forming, landscape-dominant seaweeds, and we were interested in their effects on the underlying communities of meiobenthos and macrobenthos, separated in two size fractions through sieving. A new semiquantitative treatment of metabarcoding data is introduced. The results for both markers showed that the presence of the invasive seaweed had a significant effect on the understory communities for Lophocladia lallemandii and Asparagopsis armata but not for Caulerpa cylindracea. Likewise, changes in MOTU richness and diversity with invasion status varied in magnitude and direction depending on the alga considered. Our results showed that metabarcoding allows monitoring of the less conspicuous, but not least important, effects of the presence of dominant invasive seaweeds.

The unintentional transport of invasive species through the global shipping network causes substantial losses to social and economic welfare. Addressing this global challenge requires identification of potentially harmful species, and confirmation of their movement along highly frequented shipping routes.
As we have previously shown, properly calibrated network models are able to describe passive movement of invasive species around the world. These models can be substantially improved when suitable in-situ biological data is becoming available, now possible by sequencing of environmental DNA (eDNA) from port waters.
Here we report a simple and scalable approach to generate metabarcoding data of 18S ribosomal and other eDNA collected in four major US ports. Between Long Beach, Houston, Miami, Baltimore and a multitude of Chinese ports, ships travel both frequently or infrequently while linking to different ecosystems of East Asia.
By controlling for ecoregions and ship traffic, we will shortly be able to estimate ship-borne invasive species spread between the two largest global economies, USA and China. With further port DNA sampling and network model refinements, we will also soon be able to provide global assessments of ship-borne invasive species spread to inform management and policy decision makers.

Tuesday, February 27, 2018

Anole evolution

Green Anole (Anulis carolinensis)
Squamates include all lizards and snakes, and display some of the most diverse and extreme morphological adaptations among vertebrates. However, compared with birds and mammals, relatively few resources exist for comparative genomic analyses of squamates, hampering efforts to understand the molecular bases of phenotypic diversification in such a speciose clade. In particular, the ∼400 species of anole lizard represent an extensive squamate radiation. 

Spreading through the Americas, the anoles lizards, evolved like Darwin's finches, adapting to different islands and different habitats on the mainland resulting in more than 400 species. Colleagues at the Smithsonian Tropical Research Institute (STRI) and Arizona State University compared anole lizard genomes searching for genetic evidence for rapid evolution that may account for differences between bodies of animals living in different environments. 

They discovered that changes in genes involved in the diencephalon (consisting of the hypothalamus, thalamus, metathalamus, pineal glands, and epithalamus), for color vision, hormones and the colorful dewlap that males bob to attract females, may contribute to the formation of boundaries between species. Genes regulating limb development also evolved especially quickly. One obvious explanation for such a faster rate of evolution is the anole lizards' faster rate of reproduction. Anoles typically mate in their first year of life, while other reptiles take much longer to reach sexual maturity. They also breed with many other individuals so mutations that make it difficult for individuals to survive are eliminated fairly quickly.

Overall, it seems that molecular changes underlying behavioral adaptations known to reinforce species boundaries were a key component in the diversification of anole lizards.

Friday, February 23, 2018

Weekend reads

And another load of DNA barcoding must-reads right on time for the weekend.

Understanding feedback between above- and below-ground processes of biological communities is a key to the effective management of natural and agricultural ecosystems. However, as above- and below-ground food webs are often studied separately, our knowledge of material flow and community dynamics in terrestrial ecosystems remains limited.
We developed a high-throughput sequencing method for examining how spiders link above- and below-ground food webs as generalist predators. To overcome problems related to DNA-barcoding-based analyses of arthropod-arthropod interactions, we designed spider-specific blocking primers and Hexapoda-specific primers for the selective PCR amplification of Hexapoda prey sequences from spider samples. By applying the new DNA metabarcoding framework to spider samples collected in a temperate secondary forest in Japan, we explored the structure of a food web involving 15 spider species and various taxonomic groups of Hexapoda prey. These results support the hypothesis that multiple spider species in a community can prey on both above- and below-ground prey species, potentially coupling above- and below-ground food-web dynamics.
The PCR primers and metabarcoding pipeline described in this study are expected to accelerate nuclear marker-based analyses of food webs, illuminating poorly understood trophic interactions in ecosystems.

Different second-generation sequencing technologies may have taxon-specific biases when DNA metabarcoding prey in predator feces. Our major objective was to examine differences in prey recovery from bat guano across two different sequencing workflows using the same fecal DNA extracts. We compared results between the Ion Torrent PGM and the Illumina MiSeq with similar library preparations and the same analysis pipeline. We focus on repeatability and provide an R Notebook in an effort toward transparency for future methodological improvements. Full documentation of each step enhances the accessibility of our analysis pipeline. We tagged DNA from insectivorous bat fecal samples, targeted the arthropod cytochrome c oxidase I (COI) minibarcode region, and sequenced the product on both second-generation sequencing platforms. We developed an analysis pipeline with a high operational taxonomic unit (OTU) clustering threshold (i.e., ≥ 98.5%) followed by copy number filtering to avoid merging rare but genetically similar prey into the same OTUs. With this workflow, we detected 297 unique prey taxa, of which 74% were identified at the species-level. Of these, 104 (35%) prey OTUs were detected by both platforms, 176 (59%) OTUs were detected by the Illumina MiSeq system only, and 17 (6%) OTUs were detected using the Ion Torrent system only. Costs were similar between platforms but the Illumina MiSeq recovered six times more reads and four additional insect orders than did Ion Torrent. The considerations we outline are particularly important for long-term ecological monitoring; a more standardized approach will facilitate comparisons between studies and allow faster recognition of changes within ecological communities.

The recent recolonization of Central Europe by the European gray wolf (Canis lupus) provides an opportunity to study the dynamics of parasite transmission for cases when a definitive host returns after a phase of local extinction. We investigated whether a newly established wolf population increased the prevalence of those parasites in ungulate intermediate hosts representing wolf prey, whether some parasite species are particularly well adapted to wolves, and the potential basis for such adaptations. We recorded Sarcocystis species richness in wolves and Sarcocystis prevalence in ungulates harvested in study sites with and without permanent wolf presence in Germany using microscopy and DNA metabarcoding. Sarcocystis prevalence in red deer (Cervus elaphus) was significantly higher in wolf areas (79.7%) than in control areas (26.3%) but not in roe deer (Capreolus capreolus) (97.2% vs. 90.4%) or wild boar (Sus scrofa) (82.8% vs. 64.9%). Of 11 Sarcocystis species, Sarcocystis taeniata and Sarcocystis grueneri occurred more often in wolves than expected from the Sarcocystis infection patterns of ungulate prey. Both Sarcocystis species showed a higher increase in prevalence in ungulates in wolf areas than other Sarcocystis species, suggesting that they are particularly well adapted to wolves, and are examples of "wolf specialists". Sarcocystis species richness in wolves was significantly higher in pups than in adults. "Wolf specialists" persisted during wolf maturation. The results of this study demonstrate that (1) predator-prey interactions influence parasite prevalence, if both predator and prey are part of the parasite life cycle, (2) mesopredators do not necessarily replace the apex predator in parasite transmission dynamics for particular parasites of which the apex predator is the definitive host, even if meso- and apex predators were from the same taxonomic family (here: Canidae, e.g., red foxes Vulpes vulpes), and (3) age-dependent immune maturation contributes to the control of protozoan infection in wolves.

Cephalopods are primarily active predators throughout life. Flying squids (family Ommastrephidae) represents the most widely distributed and ecologically important family of cephalopods. While the diets of adult flying squids have been extensively studied, the first feeding diet of early paralarvae remains a mystery. The morphology of this ontogenetic stage notably differs from other cephalopod paralarvae, suggesting a different feeding strategy. Here, a combination of Laser Capture Microdissection (LCM) and DNA metabarcoding of wild-collected paralarvae gut contents for eukaryotic 18S v9 and prokaryotic 16S rRNA was applied, covering almost every life domain. The gut contents were mainly composed by fungus, plants, algae and animals of marine and terrestrial origin, as well as eukaryotic and prokaryotic microorganisms commonly found in fecal pellets and particulate organic matter. This assemblage of gut contents is consistent with a diet based on detritus. The ontogenetic shift of diet from detritivore suspension feeding to active predation represents a unique life strategy among cephalopods and allows ommastrephid squids to take advantage of an almost ubiquitous and accessible food resource during their early stages. LCM was successfully applied for the first time to tiny, wild-collected marine organisms, proving its utility in combination with DNA metabarcoding for dietary studies.