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.

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.

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.