No recovery in the biomass of flying insects over the last decade in German nature protected areas.

Five years after a German study on insect biomass described a multi-decade decline in nature protected habitats, the DINA (Diversity of Insects in Nature protected Areas) project has investigated the status of insects in 21 selected nature reserves across Germany in the years 2020 and 2021. We used the same methods and protocols for trapping and measuring the biomass of flying insects as in the earlier study. Across two vegetation periods, we accumulated a comprehensive data set of 1621 data points of two-week emptying intervals to evaluate the insect biomass along gradients from arable land into nature reserves through transects of Malaise traps. On average, we observed an increase in maximum insect biomass per day along the transect from the edge to the centre of the nature reserve. Overall, the measured insect biomass remained at low levels, consistent with previous findings from the years 2007-2016. There were no significant regional differences. The results show that protected habitats have higher insect biomass compared to farmland and are therefore essential for insects but are unlikely to be sufficient to sustain insect biodiversity. Further measures need to be taken for better protection and sustainment of insects, which fulfil key functions in all terrestrial ecosystems.

Repeated subsamples during DNA extraction reveal increased diversity estimates in DNA metabarcoding of Malaise traps.

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.

Direct pesticide exposure of insects in nature conservation areas in Germany.

In Germany, the decline of insect biomass was observed in nature conservation areas in agricultural landscapes. One of the main causal factors discussed is the use of synthetic pesticides in conventional agriculture. In a Germany-wide field study, we collected flying insects using Malaise traps in nature conservation areas adjacent to agricultural land. We used a multi-component chemical trace element analysis to detect 92 common agricultural pesticides in ethanol from insect traps sampled in May and August 2020. In total, residues of 47 current use pesticides were detected, and insect samples were on average contaminated with 16.7 pesticides. Residues of the herbicides metolachlor-S, prosulfocarb and terbuthylazine, and the fungicides azoxystrobin and fluopyram were recorded at all sites. The neonicotinoid thiacloprid was detected in 16 of 21 nature conservation areas, most likely due to final use before an EU-wide ban. A change in residue mixture composition was noticeable due to higher herbicide use in spring and increasing fungicide applications in summer. The number of substances of recorded residues is related to the proportion of agricultural production area in a radius of 2000 m. Therefore, a drastic pesticide reduction in large buffers around nature conservation areas is necessary to avoid contamination of their insect fauna.

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.

More than 75 percent decline over 27 years in total flying insect biomass in protected areas.

Global declines in insects have sparked wide interest among scientists, politicians, and the general public. Loss of insect diversity and abundance is expected to provoke cascading effects on food webs and to jeopardize ecosystem services. Our understanding of the extent and underlying causes of this decline is based on the abundance of single species or taxonomic groups only, rather than changes in insect biomass which is more relevant for ecological functioning. Here, we used a standardized protocol to measure total insect biomass using Malaise traps, deployed over 27 years in 63 nature protection areas in Germany (96 unique location-year combinations) to infer on the status and trend of local entomofauna. Our analysis estimates a seasonal decline of 76%, and mid-summer decline of 82% in flying insect biomass over the 27 years of study. We show that this decline is apparent regardless of habitat type, while changes in weather, land use, and habitat characteristics cannot explain this overall decline. This yet unrecognized loss of insect biomass must be taken into account in evaluating declines in abundance of species depending on insects as a food source, and ecosystem functioning in the European landscape.