Environmental DNA from archived leaves reveals widespread temporal turnover and biotic homogenization in forest arthropod communities.

A major limitation of current reports on insect declines is the lack of standardized, long-term, and taxonomically broad time series. Here, we demonstrate the utility of environmental DNA from archived leaf material to characterize plant-associated arthropod communities. We base our work on several multi-decadal leaf time series from tree canopies in four land use types, which were sampled as part of a long-term environmental monitoring program across Germany. Using these highly standardized and well-preserved samples, we analyze temporal changes in communities of several thousand arthropod species belonging to 23 orders using metabarcoding and quantitative PCR. Our data do not support widespread declines of α-diversity or genetic variation within sites. Instead, we find a gradual community turnover, which results in temporal and spatial biotic homogenization, across all land use types and all arthropod orders. Our results suggest that insect decline is more complex than mere α-diversity loss, but can be driven by ß-diversity decay across space and time.

Insects are a barometer of environmental health. Ecosystems around the world are being subjected to unprecedented man-made stresses, ranging from climate change to pollution and intensive land use. These stresses have been associated with several recent, dramatic declines in insect populations, particularly in areas with heavily industrialised farming practices. Despite this, the links between insect decline, environmental stress, and ecosystem health are still poorly-understood. A decline in one area might look catastrophic, but could simply be part of normal, longer-term variations. Often, we do not know whether insect decline is a local phenomenon or reflects wider environmental trends. Additionally, most studies do not go far back enough in time or cover a wide enough geographical range to make these distinctions. To understand and combat insect decline, we therefore need reliable methods to monitor insect populations over long periods of time. To solve this problem, Krehenwinkel, Weber et al. gathered data on insect communities from a new source: tree leaves. Originally, these samples were collected to study air pollution, but they also happen to contain the DNA of insects that interacted with them before they were collected ­ for example, DNA deposited in chew marks where the insects had nibbled on the leaves. This is called environmental DNA, or eDNA for short. To survey the insect communities that lived in these trees, Krehenwinkel, Weber et al. first extracted eDNA from the leaves and sequenced it. Analysis of the different DNA sequences from the leaf samples revealed not only the number of insect species, but also the abundance (or rarity) of each species within each community. Importantly, the leaves had been collected and stored in stable conditions over several decades, allowing changes in these insect populations to be tracked over time. eDNA analysis revealed subtle changes in the make-up of forest insect communities. In the forests where the leaves were collected, the total number of insect species remained much the same over time. However, many individual species still declined, only to be replaced by newcomer species. These 'colonisers' are also widespread, which will likely lead to an overall pattern of fewer species that are more widely distributed ­ in other words, more homogeneity. The approach of Krehenwinkel, Weber et al. provides a reliable method to study insect populations in detail, over multiple decades, using archived samples from environmental studies. The information gained from this has real-world significance for environmental issues with enormous social impact, ranging from conservation, to agriculture and even public health.

Analysis of pesticide and persistent organic pollutant residues in German bats.

Bats are strictly protected throughout Europe. They are a highly diverse order of mammals in terms of body size, body weight, migratory behaviour, trophic niche specialisation and habitat use. The latter ranges from urban areas and arable land to forest. Due to their low reproductive rate, environmental stressors can have a major impact on bat populations. Pesticides in particular are discussed as an important driver of bat population declines. In this work, we analysed nearly 400 animals of five different species (Eptesicus serotinus, Myotis myotis, Nyctalus noctula, Pipistrellus pipistrellus, and Plecotus auritus) from all over Germany for residues of 209 pesticides and persistent organic pollutants. Residue analysis was conducted with a previously developed method using a miniaturized quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation and gas chromatography-tandem mass spectrometry for separation and detection. These analytical data were statistically correlated with the known data on the animals (e.g. age, sex, place and time of finding). Of 209 pesticides and pollutants investigated, 28 compounds were detected, the most frequent being organochlorine insecticides and polychlorinated biphenyls, which have been banned for decades by the Stockholm Convention on Persistent Organic Pollutants. Detection of more recent pesticides that were legally used for the last decade included azole antifungals and the insecticide fipronil. The bats contained between four and 25 different residues. Statistical data analyses showed that the distribution throughout Germany is largely comparable, and single exceptions were observed in specialized ecological niches. In conclusion, this work provides the largest dataset of pesticide and persistent organic pollutant residues in European bats to date.

Miniaturized multiresidue method for the analysis of pesticides and persistent organic pollutants in non-target wildlife animal liver tissues using GC-MS/MS.

In order to gain a better insight into pesticide and pollutant exposure of small (non-target) wildlife animals, a QuEChERS sample preparation method was first developed for 5 g liver tissues (e.g. hedgehog samples) and then downscaled for the analysis of 100 mg liver tissues (e.g. bat samples). The optimized (micro) QuEChERS methods used 1% acetic acid in acetonitrile as organic solvent for liquid-liquid extraction (LLE) and salting out was performed with anhydrous magnesium sulfate and sodium acetate (4:1). After a freezing-out step, sample clean-up was carried out with anhydrous magnesium sulfate, PSA, C18, and GCB (150:25:20:5). Overall, 209 pesticides and persistent organic pollutants (POPs) can be analysed within each sample with gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). Both methods were validated with representative analytes according to the European Commission guideline SANTE/12682/2019. Limits of quantification were between 1 and 20 µg kg-1, and the methods proved to be linear up to 400 µg kg-1. Additionally, the analytes delivered satisfactory results regarding recovery and precision. As proof of concept, samples of six hedgehog livers were analysed with both methods to prove the accuracy of the micro QuEChERS method. Additionally, six livers of different bat species were analysed with the downscaled method. The newly developed micro QuEChERS method for multiresidue analysis requires only minute amounts of biomaterial and represents a sophisticated novel technique for determining the exposure of small wildlife animals to different contaminants.

Determination of multi pesticide residues in leaf and needle samples using a modified QuEChERS approach and gas chromatography-tandem mass spectrometry.

In order to gain a better insight into pesticide and pollutant exposure in forests, a rapid and sensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) method for the determination of 208 pesticide residues in leaves and needles has been established. The modified QuEChERS (quick, easy, cheap, effective, rugged and safe) approach uses 2 g of homogenized sample, acetonitrile and water as extraction agents, combined with citrate buffer for the following salting out step. The limits of quantification (LOQs) were determined to 0.0025-0.05 mg kg-1, respectively. Calibration curves showed a linear range between the respective LOQ and 1.0 mg kg-1 with coefficients of determination (R2) ≥ 0.99 for all analyzed pesticides. The recovery rates ranged from 69.7% to 92.0% with a relative standard deviation below 20%. The analysis of beech leaves, spruce and pine needles (each n = 3) provided a proof of concept for the developed methodology and revealed the presence of six pesticide residues (boscalid, epoxiconazole, fenpropimorph, lindane, terbuthylazine, terbuthylazine-desethyl). The results underline the strong need for systematic surveillance of the uncontrollable exposure of pesticides to nature.