Development and evaluation of fish eDNA metabarcoding assays facilitate the detection of cryptic seahorse taxa (family: Syngnathidae)

Abstract

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Under the karst: detecting hidden subterranean assemblages using eDNA metabarcoding in the caves of Christmas Island, Australia

“…Filtration controls and extraction blanks, containing no sample, were extracted and processed alongside all samples in order to detect any cross-contamination introduced from the laboratory environment. DNA was amplified using three previously published PCR assays [27][28][29] to largely target bony fish, molluscs and arthropods (such as crustaceans and insects) from our mixed environmental samples (see Table 2 for details). Quantitative PCR (qPCR) amplification was performed using fusion tagged primers that consist of an Illumina sequencing adaptor, a unique multiplexing index (8 bp in length) and a primer sequence from each respective assay.…”

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

“…7b, c), and modified primers to accommodate their unique variations effectively increase their detections (M. Miya, unpublished data). More recently, a similar example was reported for cryptic seahorse taxa (family Syngnathidae) from Perth, Australia, in which false negatives were apparent with the existing primer sets, including MiFish, although the newly developed primers could detect these sea horse taxa (Nester et al 2020). This finding is, however, in contrast with the consistent detections of two species of seahorse in the UK using MiFish primers (Tang et al 2018).…”

“…pointed out that there were several methodological challenges that must be addressed before the eDNA metabarcoding approach is likely to become a mainstream technology in fish biodiversity research. One of these challenges is completeness and accuracy of the reference sequence database, which is currently far from satisfactory considering the enormous diversity of fish encompassing more than 32,000 species known from aquatic environments worldwide (Nelson et al 2016). Indeed, the custom reference sequence database in our laboratory (named "MiFish DB") included 5085 MiFish sequences, covering around 4230 fish species placed in 457 families and 1827 genera, as of 4 October 2014, when the original study was published .…”

“…In contrast, the enormous biodiversity in aquatic ecosystems, which include a myriad of unknown species, cannot automatically be monitored like physical attributes, making continuous biodiversity monitoring difficult (Fujikura et al 2010;DiBattista et al 2017;Alexander et al 2019). Regarding fish, over 32,000 species are known from aquatic environments worldwide, and an average of around 400 new species were described annually between 2005(Nelson et al 2016. Moreover, biodiversity monitoring of macroorganisms such as fishery resources is laborious, costly, and time consuming, often relying on direct capture of specimens through various kinds of netting, trapping, or fishing (Thomsen et al 2012;Evans et al 2017;Oka et al 2020).…”

“…Weigand et al (2019) implicitly stated, "the 12S marker has a high potential to become the gold standard for regular eDNA-based fish monitoring in the future." To date, the UK and Swedish governmental agencies have commissioned environmental survey projects using eDNA metabarcoding of fishes, and two private environmental research firms have conducted the surveys using MiFish eDNA metabarcoding (Tang et al 2018;Hellström and Blomstrand 2019;Näslund et al 2019;Tang 2020). The second initiative involves several universities and research institutes in the California Current region in a joint initiative to consider using MiFish eDNA metabarcoding to supplement traditional capturebased sampling to improve the efficiency of biodiversity monitoring in that region (Kacev et al 2018).…”