Future of DNA-based insect monitoring.

Insects are crucial for ecosystem health but climate change and pesticide use are driving massive insect decline. To mitigate this loss, we need new and effective monitoring techniques. Over the past decade there has been a shift to DNA-based techniques. We describe key emerging techniques for sample collection. We suggest that the selection of tools should be broadened, and that DNA-based insect monitoring data need to be integrated more rapidly into policymaking. We argue that there are four key areas for advancement, including the generation of more complete DNA barcode databases to interpret molecular data, standardisation of molecular methods, scaling up of monitoring efforts, and integrating molecular tools with other technologies that allow continuous, passive monitoring based on images and/or laser imaging, detection, and ranging (LIDAR).

Comparison of destructive and nondestructive DNA extraction methods for the metabarcoding of arthropod bulk samples.

DNA metabarcoding is routinely used for biodiversity assessment, in particular targeting highly diverse groups for which limited taxonomic expertise is available. Various protocols are currently in use, although standardization is key to its application in large-scale monitoring. DNA metabarcoding of arthropod bulk samples can be conducted either destructively from sample tissue, or nondestructively from sample fixative or lysis buffer. Nondestructive methods are highly desirable for the preservation of sample integrity but have yet to be experimentally evaluated in detail. Here, we compare diversity estimates from 14 size-sorted Malaise trap samples processed consecutively with three nondestructive approaches (one using fixative ethanol and two using lysis buffers) and one destructive approach (using homogenized tissue). Extraction from commercial lysis buffer yielded comparable species richness and high overlap in species composition to the ground tissue extracts. A significantly divergent community was detected from preservative ethanol-based DNA extraction. No consistent trend in species richness was found with increasing incubation time in lysis buffer. These results indicate that nondestructive DNA extraction from incubation in lysis buffer could provide a comparable alternative to destructive approaches with the added advantage of preserving the specimens for postmetabarcoding taxonomic work but at a higher cost per sample.

Metabarcoding dietary analysis in the insectivorous bat Nyctalusleisleri and implications for conservation.

In this study, we aim to uncover diet preferences for the insectivorous bat Nyctalusleisleri (Leisler's bat, the lesser noctule) and to provide recommendations for conservation of the species, based on the analysis of prey source habitats. Using a novel guano trap, we sampled bat faeces at selected roosts in a forest in Germany and tested two mitochondrial markers (COI and 16S) and three primer pairs for the metabarcoding of bat faecal pellets. We found a total of 17 arthropod prey orders comprising 358 species in N.leisleri guano. The most diverse orders were Lepidoptera (126 species), Diptera (86 species) and Coleoptera (48 species), followed by Hemiptera (28 species), Trichoptera (16 species), Neuroptera (15 species) and Ephemeroptera (10 species), with Lepidoptera species dominating in spring and Diptera in summer. Based on the ecological requirements of the most abundant arthropod species found in the bat guano, we propose some recommendations for the conservation of N.leisleri that are relevant for other insectivorous bat species.

Pooling size sorted Malaise trap fractions to maximize taxon recovery with metabarcoding.

BACKGROUND: Small and rare specimens can remain undetected when metabarcoding is applied on bulk samples with a high specimen size heterogeneity. This is especially critical for Malaise trap samples, where most of the biodiversity is contributed by small taxa with low biomass. The separation of samples in different size fractions for downstream analysis is one possibility to increase detection of small and rare taxa. However, experiments systematically testing different size sorting approaches and subsequent proportional pooling of fractions are lacking, but would provide important information for the optimization of metabarcoding protocols. We set out to find a size sorting strategy for Malaise trap samples that maximizes taxonomic recovery but remains scalable and time efficient. METHODS: Three Malaise trap samples were sorted into four size classes using dry sieving. Each fraction was homogenized and lysed. The corresponding lysates were pooled to simulate unsorted samples. Pooling was additionally conducted in equal proportions and in four different proportions enriching the small size fraction of samples. DNA from the individual size classes as well as the pooled fractions was extracted and metabarcoded using the FwhF2 and Fol-degen-rev primer set. Additionally, alternative wet sieving strategies were explored. RESULTS: The small size fractions harboured the highest diversity and were best represented when pooling in favour of small specimens. Metabarcoding of unsorted samples decreases taxon recovery compared to size sorted samples. A size separation into only two fractions (below 4 mm and above) can double taxon recovery compared to not size sorting. However, increasing the sequencing depth 3- to 4-fold can also increase taxon recovery to levels comparable with size sorting, but remains biased towards biomass rich taxa in the sample. CONCLUSION: We demonstrate that size fractionation of Malaise trap bulk samples can increase taxon recovery. While results show distinct patterns, the lack of statistical support due to the limited number of samples processed is a limitation. Due to increased speed and lower risk of cross-contamination as well as specimen damage we recommend wet sieving and proportional pooling of the lysates in favour of the small size fraction (80-90% volume). However, for large-scale projects with time constraints, increasing sequencing depth is an alternative solution.

Metabarcoding Malaise traps and soil eDNA reveals seasonal and local arthropod diversity shifts.

Forest habitats host enormous diversity, but little is known about the seasonal turnover of arthropod species between the above- and below ground forest layers. In this study, we used metabarcoding approaches to uncover arthropod diversity in different forest types and seasons. Our study shows that metabarcoding soil eDNA and Malaise trap bulk samples can provide valuable insights into the phenology and life cycles of arthropods. We found major differences in arthropod species diversity between soil samples and Malaise traps, with only 11.8% species overlap. Higher diversity levels were found in Malaise traps in summer whereas soil samples showed a diversity peak in winter, highlighting the seasonal habitat preferences and life strategies of arthropods. We conclude that collecting time series of bulk arthropod samples and eDNA in the same locations provides a more complete picture of local arthropod diversity and turnover rates and may provide valuable information on climate induced phenological shifts for long-term monitoring.

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes.

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence-capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence-capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence-capture recovered 80%-90% of the control sample species. DNA sequence-capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low-cost, whereas DNA-sequence capture for biodiversity assessment is still in its infancy, is more time-consuming but provides more taxonomic assignments.

Exploring the taxonomic composition of two fungal communities on the Swedish west coast through metabarcoding.

BACKGROUND: Fungi are heterotrophic, unicellular or filamentous organisms that exhibit a wide range of different lifestyles as, e.g., symbionts, parasites, and saprotrophs. Mycologists have traditionally considered fungi to be a nearly exclusively terrestrial group of organisms, but it is now known that fungi have a significant presence in aquatic environments as well. We know little about most fungi in limnic and marine systems, including aspects of their taxonomy, ecology, and geographic distribution. The present study seeks to improve our knowledge of fungi in the marine environment. The fungal communities of two coastal marine environments of the Kattegat sea, Sweden, were explored with metabarcoding techniques using the nuclear ribosomal internal transcribed spacer 2 (ITS2) metabarcode. Our data add new information to the current picture of fungal community composition in benthic and coastal habitats in Northern Europe. NEW INFORMATION: The dataset describes the number of operational taxonomic units (OTUs) and their taxonomic affiliations in two littoral gradients sampled on the Swedish west coast, Gothenburg municipality. Our data include basic diversity indices as well as chemical and edaphic sediment/soil parameters of the sampling sites. From the sites, 3470 and 4315 fungal OTUs, respectively, were recovered. The number of reads were 673,711 and 779,899, respectively, after quality filtering. Within the benthic sites, more than 80% of the sequences could not be classified taxonomically. The phylum composition of the classifiable sequences was dominated in both localities by Dikarya, which made up around 33% of the OTUs. Within Dikarya, Ascomycota was the dominant phylum. Guild assignment failed for more than half of the classifiable OTUs, with undefined saprotrophs being the most common resolved guild. This guild classification was slightly more common in the ocean sediment samples than in the terrestrial ones. Our metadata indicated that ocean sites contain organisms at a lower trophic level and that there are predominantly endophytic, parasitic, and pathogenic fungi in the marine environments. This hints at the presence of interesting and currently poorly understood fungus-driven ecological processes. It is also clear from our results that a very large number of marine fungi are in urgent need of taxonomic study and formal description.

DNA metabarcoding reveals diverse diet of the three-spined stickleback in a coastal ecosystem.

The three-spined stickleback (Gasterosteus aculeatus L., hereafter 'stickleback') is a common mesopredatory fish in marine, coastal and freshwater areas. In large parts of the Baltic Sea, stickleback densities have increased >10-fold during the last decades, and it is now one of the dominating fish species both in terms of biomass and effects on lower trophic levels. Still, relatively little is known about its diet-knowledge which is essential to understand the increasing role sticklebacks play in the ecosystem. Fish diet analyses typically rely on visual identification of stomach contents, a labour-intensive method that is made difficult by prey digestion and requires expert taxonomic knowledge. However, advances in DNA-based metabarcoding methods promise a simultaneous identification of most prey items, even from semi-digested tissue. Here, we studied the diet of stickleback from the western Baltic Sea coast using both DNA metabarcoding and visual analysis of stomach contents. Using the cytochrome oxidase (CO1) marker we identified 120 prey taxa in the diet, belonging to 15 phyla, 83 genera and 84 species. Compared to previous studies, this is an unusually high prey diversity. Chironomids, cladocerans and harpacticoids were dominating prey items. Large sticklebacks were found to feed more on benthic prey, such as amphipods, gastropods and isopods. DNA metabarcoding gave much higher taxonomic resolution (median rank genus) than visual analysis (median rank order), and many taxa identified using barcoding could not have been identified visually. However, a few taxa identified by visual inspection were not revealed by barcoding. In summary, our results suggest that the three-spined stickleback feeds on a wide variety of both pelagic and benthic organisms, indicating that the strong increase in stickleback populations may affect many parts of the Baltic Sea coastal ecosystem.

Taxonomy assignment approach determines the efficiency of identification of OTUs in marine nematodes.

Precision and reliability of barcode-based biodiversity assessment can be affected at several steps during acquisition and analysis of data. Identification of operational taxonomic units (OTUs) is one of the crucial steps in the process and can be accomplished using several different approaches, namely, alignment-based, probabilistic, tree-based and phylogeny-based. The number of identified sequences in the reference databases affects the precision of identification. This paper compares the identification of marine nematode OTUs using alignment-based, tree-based and phylogeny-based approaches. Because the nematode reference dataset is limited in its taxonomic scope, OTUs can only be assigned to higher taxonomic categories, families. The phylogeny-based approach using the evolutionary placement algorithm provided the largest number of positively assigned OTUs and was least affected by erroneous sequences and limitations of reference data, compared to alignment-based and tree-based approaches.

BioVeL: a virtual laboratory for data analysis and modelling in biodiversity science and ecology.

BACKGROUND: Making forecasts about biodiversity and giving support to policy relies increasingly on large collections of data held electronically, and on substantial computational capability and capacity to analyse, model, simulate and predict using such data. However, the physically distributed nature of data resources and of expertise in advanced analytical tools creates many challenges for the modern scientist. Across the wider biological sciences, presenting such capabilities on the Internet (as "Web services") and using scientific workflow systems to compose them for particular tasks is a practical way to carry out robust "in silico" science. However, use of this approach in biodiversity science and ecology has thus far been quite limited. RESULTS: BioVeL is a virtual laboratory for data analysis and modelling in biodiversity science and ecology, freely accessible via the Internet. BioVeL includes functions for accessing and analysing data through curated Web services; for performing complex in silico analysis through exposure of R programs, workflows, and batch processing functions; for on-line collaboration through sharing of workflows and workflow runs; for experiment documentation through reproducibility and repeatability; and for computational support via seamless connections to supporting computing infrastructures. We developed and improved more than 60 Web services with significant potential in many different kinds of data analysis and modelling tasks. We composed reusable workflows using these Web services, also incorporating R programs. Deploying these tools into an easy-to-use and accessible 'virtual laboratory', free via the Internet, we applied the workflows in several diverse case studies. We opened the virtual laboratory for public use and through a programme of external engagement we actively encouraged scientists and third party application and tool developers to try out the services and contribute to the activity. CONCLUSIONS: Our work shows we can deliver an operational, scalable and flexible Internet-based virtual laboratory to meet new demands for data processing and analysis in biodiversity science and ecology. In particular, we have successfully integrated existing and popular tools and practices from different scientific disciplines to be used in biodiversity and ecological research.

Preparation of Amplicon Libraries for Metabarcoding of Marine Eukaryotes Using Illumina MiSeq: The Dual-PCR Method.

This protocol details the preparation of multiplexed amplicon libraries for metabarcoding (amplicon-based) studies of microscopic marine eukaryotes. Metabarcoding studies, based on the amplification of a taxonomically informative marker from a collection of organisms or an environmental sample, can be performed to analyze biodiversity patterns or predator-prey interactions. For Metazoa, we use the mitochondrial cytochrome oxidase 1 (CO1) or the small ribosomal subunit (SSU) markers. Here, we describe a strategy for the preparation of multiplexed Illumina MiSeq libraries using a dual-PCR approach for the addition of index and adaptor sequences.

Preparation of Amplicon Libraries for Metabarcoding of Marine Eukaryotes Using Illumina MiSeq: The Adapter Ligation Method.

Amplicon-based studies of marine microscopic eukaryotes, also referred to as metabarcoding studies, can be performed to analyze patterns of biodiversity or predator-prey interactions targeting the mitochondrial cytochrome oxidase 1 (CO1) or the small ribosomal subunit (SSU) markers. Because high-throughput sequencing (HTS) Illumina platforms provide millions of reads per run, hundreds of samples may be sequenced simultaneously. This protocol details the preparation of multiplexed amplicon libraries for Illumina MiSeq sequencing. We describe a strategy for sample multiplexing using a combination of tailed PCR primers and ligation of indexed adapters.

Evaluating the potential of ecological niche modelling as a component in marine non-indigenous species risk assessments.

Marine biological invasions have increased with the development of global trading, causing the homogenization of communities and the decline of biodiversity. A main vector is ballast water exchange from shipping. This study evaluates the use of ecological niche modelling (ENM) to predict the spread of 18 non-indigenous species (NIS) along shipping routes and their potential habitat suitability (hot/cold spots) in the Baltic Sea and Northeast Atlantic. Results show that, contrary to current risk assessment methods, temperature and sea ice concentration determine habitat suitability for 61% of species, rather than salinity (11%). We show high habitat suitability for NIS in the Skagerrak and Kattegat, a transitional area for NIS entering or leaving the Baltic Sea. As many cases of NIS introduction in the marine environment are associated with shipping pathways, we explore how ENM can be used to provide valuable information on the potential spread of NIS for ballast water risk assessment.

Mapping present and future potential distribution patterns for a meso-grazer guild in the Baltic Sea.

AIM: The Baltic Sea is one of the world's largest semi-enclosed brackish water bodies characterized by many special features, including endemic species that may be particularly threatened by climate change. We mapped potential distribution patterns under present and future conditions for a community with three trophic levels. We analysed climate-induced changes in the species' distribution patterns and examined possible consequences for the chosen food web. LOCATION: Baltic Sea and northern Europe. METHODS: We developed two open-source workflow-based analytical tools: one for ecological niche modelling and another for raster layer comparison to compute the extent and intensity of change in species' potential distributions. Individual ecological niche models were generated under present conditions and then projected into a future climate change scenario (2050) for a food web consisting of a guild of meso-grazers (Idotea spp.), their host algae (Fucus vesiculosus and Fucus radicans) and their fish predator (Gasterosteus aculeatus). We used occurrence data from the Global Biodiversity Information Facility (GBIF), literature and museum collections, together with five environmental layers at a resolution of 5 and 30 arc-minutes. RESULTS: Habitat suitability for Idotea balthica and Idotea chelipes in the Baltic Sea seems to be mostly determined by temperature and ice cover rather than by salinity. 2050 predictions for all modelled species show a northern/north-eastern shift in the Baltic Sea. The distribution ranges for Idotea granulosa and G. aculeatus are predicted to become patchier in the Baltic than in the rest of northern Europe, where the species will gain more suitable habitats. MAIN CONCLUSIONS: For the Baltic Sea, climate-induced changes resulted in a gain of suitable habitats for F. vesiculosus,I. chelipes and I. balthica, whereas lower habitat suitability was predicted for I. granulosa,F. radicans and G. aculeatus. The predicted north-eastern shift of I. balthica and I. chelipes into the distribution area of F. radicans in the Baltic Sea may result in increased grazing pressure. Such additional threats to isolated Baltic populations can lead to a higher extinction risk for the species, especially as climate changes are likely to be very rapid.

Genomics in marine monitoring: new opportunities for assessing marine health status.

This viewpoint paper explores the potential of genomics technology to provide accurate, rapid, and cost efficient observations of the marine environment. The use of such approaches in next generation marine monitoring programs will help achieve the goals of marine legislation implemented world-wide. Genomic methods can yield faster results from monitoring, easier and more reliable taxonomic identification, as well as quicker and better assessment of the environmental status of marine waters. A summary of genomic methods that are ready or show high potential for integration into existing monitoring programs is provided (e.g. qPCR, SNP based methods, DNA barcoding, microarrays, metagenetics, metagenomics, transcriptomics). These approaches are mapped to existing indicators and descriptors and a series of case studies is presented to assess the cost and added value of these molecular techniques in comparison with traditional monitoring systems. Finally, guidelines and recommendations are suggested for how such methods can enter marine monitoring programs in a standardized manner.

Xenoturbella bocki exhibits direct development with similarities to Acoelomorpha.

Xenoturbella bocki, a marine animal with a simple body plan, has recently been suggested to be sister group to the Acoelomorpha, together forming the new phylum Xenacoelomorpha. The phylogenetic position of the phylum is still under debate, either as an early branching bilaterian or as a sister group to the Ambulacraria (hemichordates and echinoderms) within the deuterostomes. Although development has been described for several species of Acoelomorpha, little is known about the life cycle of Xenoturbella. Here we report the embryonic stages of Xenoturbella, and show that it is a direct developer without a feeding larval stage. This mode of development is similar to that of the acoelomorphs, supporting the newly proposed phylum Xenacoelomorpha and suggesting that the last common ancestor of the phylum might have been a direct developer.

The phylogenetic position of Acoela as revealed by the complete mitochondrial genome of Symsagittifera roscoffensis.

BACKGROUND: Acoels are simply organized unsegmented worms, lacking hindgut and anus. Several publications over recent years challenge the long-held view that acoels are early offshoots of the flatworms. Instead a basal position as sister group to all other bilaterian animals was suggested, mainly based on molecular evidence. This led to the view that features of acoels might reflect those of the last common ancestor of Bilateria, and resulted in several evo-devo studies trying to interpret bilaterian evolution using acoels as a proxy model for the "Urbilateria". RESULTS: We describe the first complete mitochondrial genome sequence of a member of the Acoela, Symsagittifera roscoffensis. Gene content and circular organization of the mitochondrial genome does not significantly differ from other bilaterian animals. However, gene order shows no similarity to any other mitochondrial genome within the Metazoa. Phylogenetic analyses of concatenated alignments of amino acid sequences from protein coding genes support a position of Acoela and Nemertodermatida as the sister group to all other Bilateria. Our data provided no support for a sister group relationship between Xenoturbellida and Acoela or Acoelomorpha. The phylogenetic position of Xenoturbella bocki as sister group to or part of the deuterostomes was also unstable. CONCLUSIONS: Our phylogenetic analysis supports the view that acoels and nemertodermatids are the earliest divergent extant lineage of Bilateria. As such they remain a valid source for seeking primitive characters present in the last common ancestor of Bilateria. Gene order of mitochondrial genomes seems to be very variable among Acoela and Nemertodermatida and the groundplan for the metazoan mitochondrial genome remains elusive. More data are needed to interpret mitochondrial genome evolution at the base of Bilateria.

The mitochondrial genome structure of Xenoturbella bocki (phylum Xenoturbellida) is ancestral within the deuterostomes.

BACKGROUND: Mitochondrial genome comparisons contribute in multiple ways when inferring animal relationships. As well as primary sequence data, rare genomic changes such as gene order, shared gene boundaries and genetic code changes, which are unlikely to have arisen through convergent evolution, are useful tools in resolving deep phylogenies. Xenoturbella bocki is a morphologically simple benthic marine worm recently found to belong among the deuterostomes. Here we present analyses comparing the Xenoturbella bocki mitochondrial gene order, genetic code and control region to those of other metazoan groups. RESULTS: The complete mitochondrial genome sequence of Xenoturbella bocki was determined. The gene order is most similar to that of the chordates and the hemichordates, indicating that this conserved mitochondrial gene order might be ancestral to the deuterostome clade. Using data from all phyla of deuterostomes, we infer the ancestral mitochondrial gene order for this clade. Using inversion and breakpoint analyses of metazoan mitochondrial genomes, we test conflicting hypotheses for the phylogenetic placement of Xenoturbella and find a closer affinity to the hemichordates than to other metazoan groups. Comparative analyses of the control region reveal similarities in the transcription initiation and termination sites and origin of replication of Xenoturbella with those of the vertebrates. Phylogenetic analyses of the mitochondrial sequence indicate a weakly supported placement as a basal deuterostome, a result that may be the effect of compositional bias. CONCLUSION: The mitochondrial genome of Xenoturbella bocki has a very conserved gene arrangement in the deuterostome group, strikingly similar to that of the hemichordates and the chordates, and thus to the ancestral deuterostome gene order. Similarity to the hemichordates in particular is suggested by inversion and breakpoint analysis. Finally, while phylogenetic analyses of the mitochondrial sequences support a basal deuterostome placement, support for this decreases with the use of more sophisticated models of sequence evolution.