Optimisation of metabarcoding for monitoring marine macrobenthos: primer choice and morphological traits determine species detection in bulkDNA and eDNA from the ethanol preservative

Abstract

DNA metabarcoding is a promising method to increase cost and time efficiency of marine monitoring, providing that the impact of methodological choices on the reliability and reproducibility of results are well understood. Here, we investigated the impact of primer choice, DNA source (bulk DNA or eDNA from the ethanol preservative) and morphological traits (body size and body skeleton) on species detection in four distinct macrobenthos communities from the North Sea. We generated a reference database with COI sequences for macrobenthos from the North sea and applied DNA metabarcoding using five COI primer sets. At most 22% of the ASVs were assigned taxonomy at the phylum level, despite the availability of a nearly complete reference database. However, the unassigned ASVs represented only a small fraction of the total reads (13%). The Leray primer set outperformed the four other primer sets in the number of non-chimeric reads and species detected, and in the recovery of beta diversity patterns. Community composition differed significantly between bulk DNA and eDNA samples, but both sample types were able to differentiate the four communities. Importantly, the probability of detecting a species in the eDNA from the ethanol preservative was significantly lower than for bulk DNA for macrobenthos species with small to medium body size and for species with chitine or CaCO3 in their skeleton. Detection in the bulk DNA samples was not affected by the traits investigated, indicating that monitoring of macrobenthos species will be most robust when using bulk DNA as template for metabarcoding.

Integration of DNA-Based Approaches in Aquatic Ecological Assessment Using Benthic Macroinvertebrates

“…Records from the search results were screened and selected for the analysis if the study targeted benthic macroinvertebrate taxa and used an (e)DNA metabarcoding approach. We found 83 papers meeting these criteria, to which seven more papers were added that met our target and were somehow missed by the search terms used [41,43,[54][55][56][57][58] (Supplementary Materials: Table S1). Papers that were not primary research articles (e.g., reviews), or targeted particular invertebrate species were excluded from the analysis.…”

“…43%) or bulk benthic communities (ca. 11%) (Figure 3), collected with sediment cores (e.g., [15,38,82,94,97]) or grabs (e.g., van Veen grab, [39,43,69,78,79,95,110]) (Table S2). Sediment sieving has been often employed to separate bulk organisms from the environmental matrix (i.e., 0.5 mm to 2 mm mesh size, e.g., [15,22,66,107]), and slicing into layers is often used to subsample marine sediments from different depths [39,87,97] (Table S2).…”

“…Regarding sediment preservation for eDNA analysis, most samples have been directly frozen (at −20 • C or −80 • C) (e.g., [38,60,61,94,95,110]), preserved in LifeGuard Soil preservation solution (MoBio, Carlsbad, CA, USA, later acquired by Qiagen, Hilden, Germany) [69,70,78,105], in ethanol at −20 • C [40,43,126], or freeze-dried [62] (Table S2). The filters' material used for water eDNA analysis (i.e., glass microfiber, cellulose nitrate, nylon, polycarbonate, or polyethersulfone), and their pore size (0.2 µm to 5 µm), have been highly variable among studies in both marine and fresh waters (e.g., [36,64,65,70,75,82,96,102,103,127]) (Table S2).…”

“…A few studies had extracted directly the DNA from preservative ethanol in order to characterize macroinvertebrate communities (e.g., [31,37,43,99,104,109]). This alternative can greatly speed up processing times in the laboratory before DNA metabarcoding because the decanting and the sorting steps can be much reduced.…”

“…Several studies have already implemented (e)DNA metabarcoding approaches to assess macroinvertebrates diversity in a wide range of aquatic ecosystems, from rivers (e.g., [24,[35][36][37]) to transitional waters (e.g., [15,27,38,39]) and coastal areas (e.g., [22,[40][41][42][43]), possibly enabling comparisons among studies and across a large temporal, spatial, and geographical scale. However, the standardization of the adopted methodologies is very difficult because a variety of factors can largely differ across studies-sampling (target community, season, effort, type of sampling devices, and site) and processing methodologies, including preservation methods and DNA extraction, PCR amplification (marker loci and primer pairs) and sequencing (platforms used) [44,45], in addition to the bioinformatics pipelines [46].…”