Pooling size sorted malaise trap fractions to maximise taxon recovery with metabarcoding

Abstract

1) Small and rare specimens can remain undetected when metabarcoding bulk samples with a high size heterogeneity of specimens. This is especially critical for malaise trap samples, where most of the biodiversity is often contributed by small specimens. How to size sort and in which proportions to pool these samples has not been widely explored. We set out to find a size sorting strategy that maximizes taxonomic recovery but remains highly scalable and time efficient. 2) Three 3 malaise trap samples where size sorted into 4 size classes using dry sieving.Each fraction was homogenized and lysed. The corresponding lysates were pooled to simulate samples never sorted, pooled in equal proportions and in 4 different proportions favoring the small size fractions. DNA from the pooled fractions as well as the individual size classes were extracted and metabarcoded using the FwhF2 and Fol-degen-rev primer set. Additionally wet sieving strategies were explored. 3) The small size fractions harbored the highest diversity, and were best represented when pooling in favor of small specimens. Not size sorting a sample leads to a 45-77% decrease in taxon recovery compared to size sorted samples. A size separation into only 2 fractions (below 4 mm and above) can already double taxon recovery compared to not sorting. However, increasing the sequencing depth 3-4 fold can also increase taxon recovery to comparable levels, but remains biased toward biomass rich taxa in the sample. 4) We demonstrate that size fractionizing bulk malaise samples can increase taxon recovery. The most practical approach is wet sieving into two size fractions, and proportional pooling of the lysates in favor of the small size fraction (80-90% volume).However, in large projects with time constraints, increasing sequencing depth can also be an alternative solution.

Recommendations for effective insect conservation in nature protected areas based on a transdisciplinary project in Germany

“…Using metabarcoding also allowed us to arrive at species lists at an unprecedented taxonomic resolution. To guarantee the best possible results for insect identification in DINA, several methodological advances like fractionization of mass bulk samples and size sorting of insects were applied [23,38,67]. Our results also show a reasonable correlation between wet biomass and insect species richness (r = 0.72, [44]).…”

Efficient assessment of nocturnal flying insect communities by combining automatic light traps and DNA metabarcoding

“…For example, Elbrecht et al. (2020) found that sorting specimens into two size fractions (above/below 4mm) doubled the number of detected species. However, this may become unpractical when hundreds of samples need to be processed, so deeper sequencing depths as the ones used in this study are probably easier to achieve.…”

Wet grinding of invertebrate bulk samples – a scalable and cost-efficient protocol for metabarcoding and metagenomics

“…Before tissue lysis, the two size fractions of the Malaise trap samples were pooled in a 1:5 ratio (large-small, 5 ml and 25 ml) as suggested by Elbrecht et al (2020). 500 µl of each sample was used for DNA extraction, which corresponds to roughly 0.1% of the complete 500 ml sample volume.…”

“…Prices may vary but go up to 15 € per sample. For sufficient homogenisation, runtime varies from 2 up to 30 mins (Elbrecht et al 2020;Beermann et al 2021). Also, sample volumes are low compared to blenders and mostly limited to 100 ml per sample.…”