New PCR primers for metabarcoding environmental DNA from freshwater eels, genus Anguilla

Abstract

Freshwater eels of the genus Anguilla comprise 16 species that include three subspecies and are characterized by their unique catadromous life cycles. Their life histories and nocturnal life styles make it difficult to observe them in freshwater and marine habitats. To investigate their distribution and ecology in aquatic environments, we developed new PCR primers for metabarcoding environmental DNA (eDNA) from Anguilla . The new primers (MiEel) were designed for two conserved regions of the mitochondrial ATP6 gene, which amplify a variable region with sufficient interspecific variations ranging from five to 22 nucleotide differences (one to three nucleotide differences between three subspecies pairs). We confirmed the versatility of the MiEel primers for all freshwater eels using tissue DNA extracts when analyzed separately. The metabarcoding combined with the MiEel primers using mock communities enabled simultaneous detection of Anguilla at the species level. Analysis of eDNA samples from aquarium tanks, a controlled pond and natural rivers demonstrated that the MiEel metabarcoding could successfully detect the correct Anguilla species from water samples. These results suggested that eDNA metabarcoding with MiEel primers would be useful for non-invasively monitoring the presence of the endangered anguillid eels in aquatic environments where sampling surveys are difficult.

A comparison of European eel Anguilla anguilla eDNA concentrations to fyke net catches in five Irish lakes

“…The primers and probe designed for this study were able to detect eDNA of A. anguilla origin in all five freshwater Irish lakes. Although the detection of A. anguilla eDNA has been previously reported in single species and metabarcoding detection studies (Seymour et al, 2018;Takeuchi et al, 2019;Thomsen, Kielgast, Iversen, Møller, et al, 2012), it was not applied for field monitoring. There is significant homology between the mtDNA of A. anguilla and A. rostrata-its and A. rostrata sourced from N. America confirmed the specificity of these primers and did not amplify using the primer-probe set (Table S1).…”

MiFish metabarcoding: a high-throughput approach for simultaneous detection of multiple fish species from environmental DNA and other samples

“…Indeed, although recent studies (Bylemans et al 2018a;Collins et al 2019;Zhang et al 2020) have demonstrated that MiFish primers outperformed other competing primers, primer/ template mismatches in MiFish primers occasionally cause underrepresentation of some fish groups (Fig. 7), and MiFish sequences often lack interspecific variations among some closely related congeners, most notably among commercially important groups, such as tunas , salmons (Morita et al 2019), and freshwater eels (Takeuchi et al 2019). However, these technical problems can be alleviated by the optimization of MiFish primers to local fauna and by simultaneous use of universal MiFish primers and newly developed group-specific primers amplifying more variable regions.…”

“…Similarly, the freshwater eel genus Anguilla also showed a lack of interspecific variations, and Takeuchi et al (2019) designed a new primer pair (MiEel) on the mitochondrial ATP6 gene with reference to the aligned mitogenome sequences from 16 constituent species. The new primer pair amplifies a variable region with sufficient interspecific variations (5-22 nucleotide differences and 1-3 nucleotide differences between three subspecies pairs), and preliminary experiments using natural waters from two rivers successfully detected Japanese freshwater eel Anguilla japonica using multiplex PCR with MiFish-U (Takeuchi et al 2019). Takeuchi et al (2019) argue that the MiEel metabarcoding would be particularly effective for documenting species distributions and for surveying spawning grounds where co-occurrence of multiple Anguilla species might be expected.…”

“…The new primer pair amplifies a variable region with sufficient interspecific variations (5-22 nucleotide differences and 1-3 nucleotide differences between three subspecies pairs), and preliminary experiments using natural waters from two rivers successfully detected Japanese freshwater eel Anguilla japonica using multiplex PCR with MiFish-U (Takeuchi et al 2019). Takeuchi et al (2019) argue that the MiEel metabarcoding would be particularly effective for documenting species distributions and for surveying spawning grounds where co-occurrence of multiple Anguilla species might be expected. Indeed, eight Anguilla A similar example can be found in some salmonid species, and a new primer pair was developed on the mitochondrial ND2 gene (Morita et al 2019).…”

“…Dots represent nucleotide sequences identical to those of the corresponding first lines. F Forward, R reverse species co-occur within and near the Indonesian archipelago and six species are known to spawn in the western South Pacific(Takeuchi et al 2019).…”