River benthic macroinvertebrates and environmental DNA metabarcoding: a scoping review of eDNA sampling, extraction, amplification and sequencing methods

Abstract

There is a growing body of literature on the use of molecular methods for the ecological assessment of rivers based on benthic macroinvertebrates. Previous research has established the benefits of the use of environmental DNA (eDNA) to assess benthic macroinvertebrate communities as being more efficient, less subjective, and non-invasive compared to traditional methods. The aim of this review is to synthesize the existing knowledge on eDNA sampling, extraction, amplification and sequencing methods regarding river benthic macroinvertebrate metabarcoding studies. Literature searches were performed using two online databases, and following a screening process, 46 papers published between 2012 and 2022 met the eligibility criteria to be included in the review. Since the use of river macrobial eDNA in ecology is a fast-evolving field, the results showed that the methodologies used vary considerably among studies. A variety of filters are used for capturing eDNA from water or preservative ethanol and different sources of eDNA (i.e., sediment, biofilm) are also explored. This review identified 12 different extraction methods and 15 different primer pairs that were used more than once in benthic macroinvertebrate eDNA metabarcoding studies. Therefore, there is a need for standardization of some key steps of the eDNA metabarcoding process to increase the comparability of the results and the robustness of the methods for further implementation into large-scale monitoring programs.

MultiBarcodeTools: Easy selection of optimal primers for eDNA multi‐metabarcoding

“…To assess the performance of MultiBarcodePipeline on non-fish species, we conducted two case studies using reference sequences of insects and fungi. In the case of insects, we obtained 19 metabarcoding primers for macroinvertebrates (Vourka et al, 2023), which comprised 15 primers targeting the cox1 gene and four primers targeting the 16S rRNA gene. The reference amplicon sequences for those 19 barcodes were constructed based on 4484 complete insect mitogenomes from the NCBI RefSeq database using the same method described in section 2.1.…”

“…The reference amplicon sequences for those 19 barcodes were constructed based on 4484 complete insect mitogenomes from the NCBI RefSeq database using the same method described in section 2.1. Then, MultiBarcodePipeline was used to recommend primer combinations in distinguishing those 4484 insect species, with the most commonly used mlCOIlintF/ jgHCO2198 (Vourka et al, 2023) being designated as the preferred primer in the parameter settings.…”

“…For insects, the most commonly used 313-bp primer barcode targeting the cox1 gene, mlCOIlintF/jgHCO2198 (Vourka et al, 2023), distinguishes 4366 insect species. Nevertheless, some closely related insects cannot be distinguished because of the biased distribution of TA B L E 3 Species identification results from MiFish amplicon sequencing data and simulated amplicon data in samples from the Sagami River, Japan (Takasaki et al, 2021).…”