Monitoring mesophotic scleractinian corals using an underwater mini-ROV to sample eDNA

Mesophotic coral ecosystems (MCEs) are light-dependent tropical or subtropical communities occurring at depths of 30 to 150 m. We recently devised a coral-specific environmental DNA (eDNA) barcoding method that can identify 36 scleractinian genera in shallow reefs by sampling ~1L of surface seawater. If eDNA barcoding is combined with sampling using underwater mini-Remote Operated Vehicles (mini-ROVs), it may be possible to survey mesophotic corals more easily and broadly. Around the Zamami Islands, in Okinawa, Japan, seawater was collected 1–2 m above the bottom at six locations 20–80 m below the surface and subjected to coral-specific eDNA amplification. Metabarcoding analyses showed that (a) eDNA from ~0.5 L seawater was sufficient to identify genera and to yield comparative ratios of genera at these sites; (b) Acropora dominates shallow reefs and upper ridges of slopes, while other genera including Porites, Pocillopora and Polyphyllia are more abundant at mesophotic sites; (c) one site showed a gradient in which Acropora was replaced by Plesiastrea at increasing depths. Although further technical improvements are required, the use of eDNA and underwater mini-ROVs may permit monitoring of mesophotic corals more broadly and easily.

Methods
Seawater samples were collected at reefs around the Zamami Islands on 9-10 March and 23-25 May 2022. Mini-ROV used in this study was FIFISH V6Plus. On the boat, seawater collected by the sampler was moved to 1L bottle and filtered promptly through 0.45-μm Sterivex filters (Merck) using peristaltic pomp. 1 mL of RNAlater (Qiagen) was added to the filtrate to prevent DNA degradation and was maintained at 4℃ before transfer to a -20℃ freezer in the laboratory. eDNA in Sterivex filters was extracted following the Environmental DNA Sampling and Experiment Manual v. 2.1. Extracted eDNA samples were PCR-amplified using primers, Scle_12S_Fw (5’-CCAGCMGACGCGGTRANACTTA-3’) and Scle_12S_Rv (5’-AAWTTGACGACGGCCATGC-3’), for mitochondrial 12S rRNA genes of scleractinian corals. PCR amplification was carried out Tks Gflex DNA Polymerase (Takara) under cycling conditions of 1 min at 94°C, followed by 35 cycles of 10 s at 98°C, 15 s at 60°C and 30 s at 68°C, with an extension of 5 min at 68°C in the final cycle. PCR products were extracted and cleaned with a FastGene Gel/PCR Extraction Kit (NIPPON Genetics Co., Ltd.). Amplicon sequencing libraries of cleaned PCR products were prepared using a KAPA Hyper Prep Kit (NIPPON Genetics) without fragmentation. Libraries were multiplexed and 300-bp paired-end reads were sequenced on a MiSeq platform (Illumina) using a MiSeq Reagent kit v3 (600 cycles).