Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes

Abstract

Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R2) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity.

How can eDNA contribute in riverine macroinvertebrate assessment? A metabarcoding approach in the Nalón River (Asturias, Northern Spain)

“…However, not all water quality indices are based on families, and the taxonomic level demanded for monitoring programs varies significantly among countries (Bonada et al, 2005). Although some studies have tried to relate the sequence reads with individual abundance metrics (Klymus, Marshall, & Stepien, 2017;Ushio et al, 2018), it is still not possible to do it with the metabarcoding analysis carried out in this study. On the other hand, quantitative elements are also currently required within the WFD.…”

Invasion genetics from eDNA and thousands of larvae: A targeted metabarcoding assay that distinguishes species and population variation of zebra and quagga mussels

“…The spacer region increased nt diversity of sequence reads and enhanced cluster formation, improving sequencing quality (Fadrosh et al, ; Wu et al, ). Four primer sets were used, which differed in their spacer combinations (see Klymus et al, ). PCR conditions were identical, except that the cycle number was reduced to eight.…”

“…Conditions were as follows: 30‐s initial denaturation at 95°C, followed by eight cycles at 95°C for 30‐s, 55°C for 30‐s, and 68°C for 1‐min, with a final 2‐min 68°C extension. Four samples were run with the Mol16S primer set followed the library preparation, which was published in Klymus et al ().…”

“…Pooled product concentrations were measured on a Qubit fluorometer (Invitrogen), and the pooled samples then were run on an Illumina ® MiSeq with 2X 300nt V3 chemistry at the Ohio State University's Molecular and Cellular Imaging Center in Wooster, OH (http://mcic.osu.edu/). An additional 40%–50% PhiX DNA spike‐in control was added to improve data quality of low nt diversity samples (Klymus et al, ). HTS was performed on four MiSeq runs, which also contained tagged samples from other projects to increase diversity; all were pooled to yield >100,000 reads per sample.…”

Responses of foraminifera communities to aquaculture‐derived organic enrichment as revealed by environmental DNA metabarcoding

“…Environmental DNA metabarcoding has been widely used for environmental assessment, such as detection of invasive species (e.g., Klymus, Marshall, & Stepien, ), water quality assessment in rivers and streams (e.g., Apothéloz‐Perret‐Gentil et al., ; Zimmermann, Glöckner, Jahn, Enke, & Gemeinholzer, ), impacts of oil drilling (e.g., Lanzén, Lekang, Jonassen, Thompson, & Troedsson, ) as well as benthic impacts of marine finfish farms (e.g., Pawlowski, Esling, Lejzerowicz, Cedhagen, & Wilding, ). Several eDNA metabarcoding studies on benthic impacts of salmon farming found results obtained using eDNA metabarcoding were comparable to those obtained using complex biological indices that require macrofaunal inventories characterized by traditional morpho‐taxonomy (Dowle, Pochon, Keeley, & Wood, ; Lejzerowicz et al., ; Pawlowski et al., , ; Pochon et al., ; Stoeck, Frühe et al., ).…”