EntoSieve: Automated Size-Sorting of Insect Bulk Samples to Aid Accurate Megabarcoding and Metabarcoding

Abstract

Widespread insect decline necessitates the development and use of standardized protocols for regular monitoring. These methods have to be rapid, efficient and cost-effective to allow for large-scale implementation. Many insect sampling and molecular methods have been developed. These include Malaise trapping, high-throughput DNA barcoding ('megabarcoding') and metabarcoding. The latter allows for assessing the species diversity in whole samples using few steps, but sample heterogeneity in terms of body size remains a challenge since large insects contribute disproportionately more mtDNA than small ones. This can potentially overwhelm the template DNA from small species that then go undetected. Size-sorting can mitigate this problem, but no satisfying automated, rapid and non-destructive solutions are available. We introduce the EntoSieve, a low-cost and DIY motorized instrument that disentangles and sorts abundant insect bulk samples into several body size fractions while minimizing damage to specimens, thus reducing the risk of DNA contamination across size fractions (e.g. legs of large specimens in small body size fraction). EntoSieve utilizes readily available components, 3D-printed parts and customizable meshes, thus enabling parallelization at low cost. We here show the efficiency of the EntoSieve for three samples with more than 10,000 specimens using three sieving protocols and assess the impact on specimen integrity. Efficiency ranged from 92% to 99%, achieved within 18-60 min, and specimen damage was not significant for subsamples. By facilitating rapid pre-processing, the device contributes to producing morphologically valuable vouchers for megabarcoding and is likely to improve compositional diversity accuracy across size classes when using metabarcoding.

Keywords: DNA barcoding; biomass bias; bulk samples; insect biodiversity; sequence recovery.

FIGURE 1

(A) EntoSieve with (1) microcontroller and rotary controls for setting speed and stroke, (2) stepper motor, (3) linear rail with spindle drive, (4) sieve container, (5) perforated sieve, (6) inner container, (7) outer container. The system is clamped (8) on the edge of a table or bench to ensure stability. The inner and outer containers stand on a (9) height‐adjustable table. (B) Two sieve containers: To quickly transfer the insects for the next size category to a second sieve container by 180° rotation on their horizontal axis, the (10) sieve containers are stackable and can be temporarily fixed in place using (11) clamps. (C) Specimens in the sieve container during sieving. (D) Inner container concept with the (12) coffee filter to separate the insects from the water.

FIGURE 2

The four size fractions separated in test 2 using meshes MA = 1.5 mm; MB = 3 mm; MC = 5 mm for 20 min each. (A) Fraction A; (B) fraction B; (C) fraction C; (D) fraction D. Pie charts illustrate the ratio of correctly sieved specimens (green) and intruders (red) of each size category. The table shows the percentage of specimens of each size category (columns) that were sorted into each fraction (rows). Numbers on columns' names indicate the total number of specimens.

FIGURE 3

The two size fractions separated in test 3 using sieve MB = 3 mm for 20 min. (A) Fraction [A + B]; (B) fraction [C + D]. Pie charts illustrate the ratio of correctly sieved specimens (green) and intruders (red) of each category. The table shows the percentage of specimens of each size category (columns) that were sorted into each fraction (rows). Numbers on columns' names indicate the total number of specimens.