MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species

TLDR

The study focuses on a taxonomically and ecologically diverse set of marine fishes ranging from sharks and rays to teleosts across shallow to deep waters. We developed universal PCR primers (MiFish‑U/E) for metabarcoding fish eDNA. Primers were designed from 880 mitogenomes and 160 elasmobranch sequences to target a 163‑185 bp hypervariable 12S rRNA region, then tested by dual‑indexed paired‑end sequencing of aquarium tank eDNA and natural reef waters. The primers detected 232 fish species (70 families, 152 genera) from aquarium tanks and reef waters, achieving a 93 % detection rate in tanks and demonstrating that the method is non‑invasive, efficient, cost‑effective, and highly sensitive, making it a promising tool for large‑scale biodiversity monitoring.

Abstract

We developed a set of universal PCR primers (MiFish-U/E) for metabarcoding environmental DNA (eDNA) from fishes. Primers were designed using aligned whole mitochondrial genome (mitogenome) sequences from 880 species, supplemented by partial mitogenome sequences from 160 elasmobranchs (sharks and rays). The primers target a hypervariable region of the 12S rRNA gene (163-185 bp), which contains sufficient information to identify fishes to taxonomic family, genus and species except for some closely related congeners. To test versatility of the primers across a diverse range of fishes, we sampled eDNA from four tanks in the Okinawa Churaumi Aquarium with known species compositions, prepared dual-indexed libraries and performed paired-end sequencing of the region using high-throughput next-generation sequencing technologies. Out of the 180 marine fish species contained in the four tanks with reference sequences in a custom database, we detected 168 species (93.3%) distributed across 59 families and 123 genera. These fishes are not only taxonomically diverse, ranging from sharks and rays to higher teleosts, but are also greatly varied in their ecology, including both pelagic and benthic species living in shallow coastal to deep waters. We also sampled natural seawaters around coral reefs near the aquarium and detected 93 fish species using this approach. Of the 93 species, 64 were not detected in the four aquarium tanks, rendering the total number of species detected to 232 (from 70 families and 152 genera). The metabarcoding approach presented here is non-invasive, more efficient, more cost-effective and more sensitive than the traditional survey methods. It has the potential to serve as an alternative (or complementary) tool for biodiversity monitoring that revolutionizes natural resource management and ecological studies of fish communities on larger spatial and temporal scales.