Understanding insect behaviour and its underlying drivers is vital for interpreting changes in local biodiversity and predicting future trends. Conventional insect traps are typically limited to assess the composition of local insect communities over longer time periods and provide only limited insights into the effects of abiotic factors, such as light on species activity. Achieving finer temporal resolution is labour-intensive or only possible under laboratory conditions. Here, we demonstrate that time-controlled insect sampling using an automated Malaise trap in combination with metabarcoding allows for the observation and documentation of taxon-specific activity patterns. Furthermore, these recorded activity patterns can provide valuable insights into the underlying ecological processes. Insect activity curves, derived from predicted detection numbers using generalised linear latent variable models, reveal distinct differences in activity patterns at higher and lower taxonomic level. While our findings align with existing literature, they also reveal that the activity patterns of some species are more complex than previously known. Additionally, a comparison of the assessed activity patterns across taxa suggest potential, previously undescribed parasitoid-host relationships. Within taxonomic groups, we observe variations in both the timing and duration of activity patterns, which can be linked to differences in mating strategies among closely related species. By capturing circadian rhythms of insect activity through time-controlled bulk sampling, we can expand our knowledge on species behaviour, ecology and temporal interactions. This contributes significantly to the advancement of chronoecology, allowing for further exploration of the roles of species and benefits in natural and anthropogenic ecosystems, alongside their potentially significant threat.

Currently, knowledge of arthropod diversity and distributions in terrestrial habitats is limited due to a lack of taxonomists and consistent monitoring, hindering insect conservation efforts. To address this, we conducted an extensive country‐wide study in Germany using a year‐long Malaise trap monitoring effort supported by citizen scientists. Our study provides a benchmark of species occurrence in 21 German nature‐protected areas. Metabarcoding methods revealed unexpectedly high small‐scale and largely undescribed diversity, with 14,445 operational taxonomic units (OTUs) found. Of these OTUs, 8,589 were assigned to a species, but only 3,064 have been evaluated in German red lists. Seventeen invasive species were identified, five of which are potentially new records for Germany. High potential regional endemism was found, with 33% of OTUs detected in only one protected area, while widespread OTUs were rare making up 1.2% of the OTUs. Six protected areas showed significantly higher local contributions to beta diversity, emphasising the importance of protecting regionally diverse habitats.

Molecular methods are currently some of the best-suited technologies for implementation in insect monitoring. However, the field is developing rapidly and lacks agreement on methodology or community standards. To apply DNA-based methods in large-scale monitoring, and to gain insight across commensurate data, we need easy-to-implement standards that improve data comparability. Here, we provide three recommendations for how to improve and harmonize efforts in biodiversity assessment and monitoring via metabarcoding: (i) we should adopt the use of synthetic spike-ins, which will act as positive controls and internal standards; (ii) we should consider using several markers through a multiplex polymerase chain reaction (PCR) approach; and (iii) we should commit to the publication and transparency of all protocol-associated metadata in a standardized fashion. For (i), we provide a ready-to-use recipe for synthetic cytochromecoxidase spike-ins, which enable between-sample comparisons. For (ii), we propose two gene regions for the implementation of multiplex PCR approaches, thereby achieving a more comprehensive community description. For (iii), we offer guidelines for transparent and unified reporting of field, wet-laboratory and dry-laboratory procedures, as a key to making comparisons between studies. Together, we feel that these three advances will result in joint quality and calibration standards rather than the current laboratory-specific proof of concepts.This article is part of the theme issue ‘Towards a toolkit for global insect biodiversity monitoring’.

Reliable biodiversity data are crucial for environmental research and management. Unfortunately, data paucity prevails for many regions and organismal groups such as aquatic invertebrates. High-throughput DNA-based identification, in particular DNA metabarcoding, has accelerated biodiversity data generation. However, in the process of metabarcoding, specimens are usually destroyed, precluding later specimen-based analyses. Metabarcoding of DNA released into the preservative ethanol has been proposed as a non-destructive alternative, but proof-of-principle studies have yielded ambiguous results, reporting variance in detection probability for various taxa and methodological biases. In this study, we tested the performance of preservative-based metabarcoding of aquatic invertebrates in comparison to a standard morpho-taxonomic assessment based on samples from one of Europe's last free-flowing rivers, the Vjosa. Multi-habitat samples were collected at 43 sites in two seasons and stored in ethanol, after fixation in formaldehyde for morpho-taxonomic analyses. Preservative-based DNA metabarcoding detected three times more taxa but failed to detect other taxa found using the standard method. In addition to incomplete reference data and primer bias that likely precluded the detection of specific taxa, preservative-based DNA metabarcoding cannot provide accurate abundance estimates. However, the metabarcoding data revealed distinct small-scale and large-scale community patterns in the Vjosa river network, which were also recovered by quantitative data of the standard approach. Overall, our results indicate that preservative-based metabarcoding provides important biodiversity data, which could be further improved by quantitative validation. The method is robust and reliable, even though samples were taken under harsh field-conditions and stored without cooling. Further, our results emphasise the need for reliable DNA barcoding reference libraries. Building those may be supported by preservative-based metabarcoding that maintains intact vouchers for subsequent specimen-based analyses.

Biodiversity is declining at alarming rates worldwide and large-scale monitoring is urgently needed to understand changes and their drivers. While classical taxonomic identification of species is time and labour intensive, the combination with DNA-based methods could upscale monitoring activities to achieve larger spatial coverage and increased sampling effort. However, challenges remain for DNA-based methods when the number of individuals per species and/or biomass estimates are required. Several methodological advancements exist to improve the potential of DNA metabarcoding for abundance analysis, which however need further evaluation. Here, we discuss laboratory, as well as some bioinformatic adjustments to DNA metabarcoding workflows regarding their potential to achieve species abundance estimation from arthropod community samples. Our review includes pre-laboratory processing methods such as specimen photography, laboratory methods such as the use of spike-in DNA as an internal standard and bioinformatic advancements like correction factors. We conclude that specimen photography coupled with DNA metabarcoding currently promises the greatest potential to achieve estimates of the number of individuals per species and biomass estimates, but that approaches such as spike-ins and correction factors are promising methods to pursue further.

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.

Most empirical metacommunity studies rely solely on morphological identification of taxa, precluding the species-level identification of several biotic groups, which can influence the characterization of metacommunities. DNA metabarcoding enables inference of species and even intraspecific diversity from community samples but has rarely been used to infer metacommunity structure. Here, we combined morphology and metabarcoding to improve the characterization of an insect metacommunity at different identification levels. We included measures of taxonomic, functional and phylogenetic richness, and we evaluated drivers affecting metacommunity structure (i.e., environmental filtering and dispersal). Communities were sampled from an area that included nine perennial, two near-perennial and two intermittent sites in a river network characterized by high hydrological variability. We identified organisms to a mixed (family to species) taxonomic level using morphology, and to operational taxonomic unit (OTU) and haplotype levels using metabarcoding of the mitochondrial cytochrome c oxidase gene. Diptera and Ephemeroptera showed the greatest increases in taxonomic and phylogenetic richness but not biological trait richness with increasing taxonomic resolution. The joint effect of environmental filtering and dispersal was more important than their individual effects in shaping metacommunity structure at all identification levels. Mixed-level and OTU-level identification were more effective than family and haplotype in characterizing the drivers of metacommunity structure. We demonstrate that the greater taxonomic resolution enabled by metabarcoding could improve understanding of metacommunities within river networks, thus enhancing our capacity to predict ecological responses in ecosystems adapting to global change.

Human-induced biodiversity loss and changes in community composition are major challenges of the present time, urgently calling for comprehensive biomonitoring approaches to understand system dynamics and to inform policy-making. In this regard, molecular methods are increasingly applied. They provide tools for fast and high-resolution biodiversity assessments and can also focus on population dynamics or functional diversity. If samples are stored under appropriate conditions, this will enable the analysis of DNA, but also RNA and proteins from tissue or from non-biological substrates such as soil, water, or sediments, so-called environmental DNA (eDNA) or eRNA. Until now, most biodiversity studies using molecular methods rely on recent sampling events, although the benefit of analyzing long-time series is obvious. In this context Environmental Specimen Banks (ESBs) can play a crucial role, supplying diverse and well-documented samples collected in periodically repeated sampling events, and following standardized protocols. Mainly assembled for integrative monitoring of chemical compounds, ESB collections are largely accessible to third parties and can in principle be used for molecular analysis. While ESBs hold great potential for the standardized long-time storage of environmental samples, the cooperation with Biodiversity Biobanks as scientific collections guarantees the long-time storage of nucleotide (DNA, RNA) extracts together with links to analytical results and metadata. The present contribution aims to raise the awareness of the biodiversity research community regarding the high-quality samples accessible through ESBs, encourages ESBs to collect and store samples in DNA-friendly ways, and points out the high potential of combining DNA-based approaches with monitoring chemicals and other environmental stressors.

In recent years, the decline of insect biodiversity and the imminent loss of provided ecosystem functions and services has received public attention and raised the demand for political action. The complex, multi-causal contributors to insect decline require a broad interdisciplinary and cross-sectoral approach that addresses ecological and social aspects to find sustainable solutions. The project Diversity of Insects in Nature protected Areas (DINA) assesses insect communities in 21 nature reserves in Germany, and considers interactions with plant diversity, pesticide exposure, spatial and climatic factors. The nature reserves border on agricultural land, to investigate impacts on insect diversity. Part of the project is to obtain scientific data from Malaise traps and their surroundings, while another part involves relevant stakeholders to identify opportunities and obstacles to insect diversity conservation. Our results indicate a positive association between insect richness and biomass. Insect richness was negatively related to the number of stationary pesticides (soil and vegetation), pesticides measured in ethanol, the amount of area in agricultural production, and precipitation. Our qualitative survey along with stakeholder interviews show that there is general support for insect conservation, while at the same time the stakeholders expressed the need for more information and data on insect biodiversity, as well as flexible policy options. We conclude that conservation management for insects in protected areas should consider a wider landscape. Local targets of conservation management will have to integrate different stakeholder perspectives. Scientifically informed stakeholder dialogues can mediate conflicts of interests, knowledge, and values to develop mutual conservation scenarios.

Due to increasing human transformation of virtually all habitats on Earth, setting global priorities for conservation is essential. The emerging disciplines of macroecology and macroevolution (MEE) can provide a global perspective and information for such prioritization but remain relatively separated from conservation prioritization, partly because MEE researchers are unaware of the requirements for effective conservation prioritization and existing approaches implementing these. Indeed, existing approaches for conservation prioritization at large scale are scattered across literature. Here, we systematically review this literature and provide a guideline for researchers in MEE as to which of approaches might be suitable for their needs. From >11 000 scientific publications, we identified 134 methods suitable for commonly used MEE data and geographic scale. Furthermore, we use a 'trait matrix' to identify families of similar approaches and to design a guideline to select the most suitable method, given a user-defined set of data and analysis scope. The guidelines are freely available via the conserveR R-package (<>). Finally, we used our review to identify increasing scalability, continuous monitoring and the integration of molecular, remote sensing and animal movement data as key areas for future impacts of MEE in conservation prioritization. We anticipate that our review can serve as a guide to MEE researchers interested in linking their data to conservation as well as for conservation scientists interested in using MEE approaches.

With increased application of DNA metabarcoding in biodiversity assessment, various laboratory protocols have been optimized, and their further evaluation is subject of current research. Homogenization of bulk samples and subsequent DNA extraction from a subsample of destructed tissue is a common first stage of the metabarcoding process. This can either be conducted using sample material soaked in a storage fixative, e.g., ethanol (here referred to as "wet" treatment) or from dried individuals ("dry"). However, it remains uncertain if perfect mixing and equal distribution of DNA within the tube is ensured during homogenization and to what extent incomplete mixing and resulting variations in tissue composition affect diversity assessments if only a fraction of the destructed sample is processed in the downstream metabarcoding workflow. Here we investigated the efficiency of homogenization under wet and dry conditions and tested how variations in destructed tissue composition might affect diversity assessments of complex arthropod samples. We considered five time intervals of Malaise trap bulk samples and process nine different subsamples of homogenized tissue (20 mg each) in both treatments. Results indicate a more consistent diversity assessment from dried material, but at the cost of a higher processing time. Both approaches detected comparable OTU diversity and revealed similar taxa compositions in a single tissue extraction. With an increased number of tissue subsamples during DNA extraction, OTU diversity increased for both approaches, especially for highly diverse samples obtained during the summer. Here, particularly the detection of small and low-biomass taxa increased. The processing of multiple subsamples in the metabarcoding protocol can therefore be a helpful procedure to enhance diversity estimates and counteract taxonomic bias in biodiversity assessments. However, the process induces higher costs and time effort and the application in large-scale biodiversity assessment, e.g., in monitoring schemes needs to be considered on project-specific prospects.

Insects are the most diverse group of animals on Earth, but their small size and high diversity have always made them challenging to study. Recent technological advances have the potential to revolutionise insect ecology and monitoring. We describe the state of the art of four technologies (computer vision, acoustic monitoring, radar, and molecular methods), and assess their advantages, current limitations, and future potential. We discuss how these technologies can adhere to modern standards of data curation and transparency, their implications for citizen science, and their potential for integration among different monitoring programmes and technologies. We argue that they provide unprecedented possibilities for insect ecology and monitoring, but it will be important to foster international standards via collaboration.

We present a unique data set of trait information for 586 insect families in Central Europe, covering the largest known part of described species (over 34,000 species). Life history information and major functional traits were evaluated with fuzzy coding and weighted according to the number of known species in Germany. An overall analysis of the German insect fauna is given and the data set is exemplarily applied to metabarcoding results of malaise trap samples. Due to the high functional and taxonomic diversity in insects, further developments and refinements of traits to be included will be an ongoing process with advancements of upcoming database versions to be subsequently published.

With increased application of DNA metabarcoding in fast and high-resolution biodiversity assessment, various laboratory protocols have been optimised in recent years and their further evaluation is subject of current research. Homogenisation of bulk samples and subsequent DNA extraction from destructed tissue is one way of starting the metabarcoding process. This essential step in the protocol can either be conducted from wet sample material (e.g. bulk insect samples) soaked in fixative or from completely dried individuals. While the latter method appears to produce more consistent results, it is time consuming and more prone to cross-contamination. We tested both homogenisation approaches with regard to time efficiency and biodiversity assessment of complex arthropod bulk samples, in particular how the amount of processed tissue affects taxon recovery. Both approaches reveal similar taxa compositions and detect a similar total OTU diversity in a single extraction reaction. Increased amounts of tissue used in DNA extraction improved OTU diversity detection and recovered particularly specific low-biomass taxa, making this approach valuable for samples with high biomass and/or diversity. Due to less handling time and lower vulnerability for cross-contamination we recommend the processing of wet material when sample homogenisation is applied.

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.

Streams and rivers represent hotspots of biodiversity in their natural state. This biodiversity is declining worldwide due to pollution, exploitation and hydromorphological degradation of these systems. One of the last big, natural rivers in Europe is the Vjosa in the Balkan region. The catchment is characterized by natural flow dynamics, resulting in high habitat diversity and turnover, and hosts several sensitive and endemic species (e.g. Isoperla vjosae). DNA metabarcoding represents a promising approach to characterize this biodiversity but methodological drawbacks such as primer bias or incomplete reference databases limit the application, particularly in taxonomically underexplored regions. Here, we assessed stream biodiversity with a focus on macrozoobenthic (MZB) taxa via a non-destructive, voucher-preserving DNA metabarcoding protocol. In this approach, ethanol used for preservation of multi-habitat samples in the field was used as DNA template, allowing to retain the integrity of the original sample and further comparison of molecular and morphological taxa lists. Samples were taken in spring and autumn 2018 at 48 sites allocated over the Vjosa catchment. The preservative ethanol was filtered through 0.43 um nitrocellulose membranes from which DNA was extracted. Subsequently, a 421 bp fragment of the COI gene was amplified with the primer pair BF2/BR2 and Illumina sequenced. After filtering for sequences with similarity to reference entries >85%, 4,123 OTUs were obtained, of which 921 were identified as MZB taxa. Dipterans and ephemeropterans were most abundant, followed by plecopterans. Some taxa (e.g. molluscs) were not identified due to a known primer bias and >7,000 OTUs could not be assigned above 85 % similarity. Using the here presented voucher-preserving approach allowed us to identify the pitfalls of DNA metabarcoding as tool for biodiversity assessment in taxonomically unexplored regions such as the Vjosa catchment. However, the comparison of specimen abundance data and molecular data showed the power of non-destructive fixative metabarcoding for detecting MZB communities with highly increased taxonomic resolution.

Multiple stressors diversely and often adversely affect stream ecosystems around the globe. Therefore, understanding multiple stressor effects on different organisms is essential for a better ecosystem understanding, an accurate water quality assessment and improved ecosystem management. However, while multiple stressor effects should be assessed at species level this taxonomic resolution is often not achieved e.g. for stream macroinvertebrates. Due to their high abundance and diversity, species-level identification is often not feasible with morphology‑based approaches. DNA metabarcoding represents an alternative approach for studying multiple stressor interactions at species level. In an outdoor experiment over 10,000 specimens from the insect orders Ephemeroptera, Plecoptera and Trichoptera (EPT), which are routinely used as bioindicators, and their responses to stressors were studied. In the experiment salinity, fine sediment deposition and flow velocity were manipulated in a full‑factorial design in 64 mesocosms with two microhabitats each (streambed and leaf litter), resulting in eight replicates per treatment. DNA metabarcoding revealed 122 EPT Operational Taxonomic Units (OTUs), from which the most abundant 27 alone showed 14 different response patterns to the applied stressors. The high taxonomic resolution achieved by DNA metabarcoding revealed species specific stressor responses that were hidden at a lower taxonomic resolution. As a prominent example, Rhithrogena semicolorata responded negatively to fine sediment deposition and flow velocity reduction, while Ecdyonurus torrentis (both Heptageniidae, Ephemeroptera) was insensitive to experimental manipulation, highlighting different stressor responses among species within the same family (Fig. 1, Beermann et al. 2020). Even for well-studied organisms such as EPT taxa, this study shows that DNA metabarcoding has the potential to depict response patterns at species or OTU level despite high specimen abundance. Consequently, DNA metabarcoding promises to be a rewarding method when investigating and assessing multiple stressor effects on stream water quality.

Insect declines and biodiversity loss have attracted much attention in recent years, but lack of comprehensive data, conflicting interests among stakeholders and insufficient policy guidance hinder progress in preserving biodiversity. The project DINA (Diversity of Insects in Nature protected Areas) investigates insect communities in 21 nature reserves in Germany. All selected conservation sites border arable land, with agricultural practices assumed to influence insect populations. We taught citizen scientists how to manage Malaise traps for insect collection, and subsequently used a DNA metabarcoding approach for species identification. Vegetation surveys, plant metabarcoding as well as geospatial and ecotoxicological analyses will help to unravel contributing factors for the deterioration of insect communities. As a pioneering research project in this field, DINA includes a transdisciplinary dialogue involving relevant stakeholders such as local authorities, policymakers, and farmers, which aims at a shared understanding of conservation goals and action pathways. Stakeholder engagement combined with scientific results will support the development of sound policy recommendations to improve legal frameworks, landscape planning, land use, and conservation strategies. With this transdisciplinary approach, we aim to provide the background knowledge to implement policy strategies that will halt further decline of insects in German protected areas.

Environmental DNA (eDNA) metabarcoding is a new, promising, and non-invasive method to detect biodiversity in aquatic environments. So far, it has mainly been used to screen for fish and amphibian diversity and rarely to detect macroinvertebrates. Typically, DNA metabarcoding relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase I (COI) gene with degenerate primers. In comparison to other genes like 16S, COI has a greater taxonomic resolution and availability of an extensive reference database. Benthic stream invertebrates are of critical importance for regulatory biomonitoring, but when using universal primers on eDNA isolated from water, the number of reads and OTUs is “watered down”. This means the target taxa, macroinvertebrates, are underrepresented in comparison to other nontarget taxa, e. g. algae, bacteria, and fungi. The aim of the project was to design an insect-specific primer, which minimizes nontarget amplification. Therefore, data from a time series of 15 months at the Kinzig (Hesse), a silica-rich low-mountain-range stream, which is part of the Rhine‑Main‑Observatory (LTER site) was generated using the universal primers BF2/BR2. With this data we identified the most abundant nontarget taxa and designed a new reverse primer (EPTDr2n) with 3’ ‐ specificity toward benthic invertebrate taxa. Primer specificity was validated in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. 20 eDNA samples from the Kinzig River and its tributaries were then used to test the new primer in situ together with primer fwhF2. The new primer combination showed a much higher amplification of benthic invertebrates, insects in particular, than two other universal primer pairs for both, number of target reads (fwhF2/EPTDr2n: 99.6% versus BF2/BR2: 25.89% and fwhF2/fwhR2n: 39.04%; Fig. 1) and number of target species (fwhF2/EPTDr2n: 305 versus BF2/BR2: 113 and fwhF2/fwhR2n: 185). Additionally, the number of benthic invertebrate species exceeded even the number of 153 species identified by expert taxonomists at nearby sites across two decades of sampling. While several taxa reported, like a few trichopteran genera, flatworms, and some crustaceans, were not found, the primer shows greatly improved results for eDNA metabarcoding of benthic invertebrates(Leese et al. 2021).