DNA metabarcoding-based diet survey for the Eurasian otter (Lutra lutra): Development of a Eurasian otter-specific blocking oligonucleotide for 12S rRNA gene sequencing for vertebrates

The Eurasian otter (Lutra lutra) is an endangered species for which diet analyses are needed as part of its conservation efforts. Eurasian otters feed on vertebrates, such as fishes, and invertebrates, such as crustaceans, but their detailed taxonomies are not fully understood in part due to limited resolving power of traditional morphological identification methods. Here, we used high-throughput sequencing (HTS)-based DNA metabarcoding approaches to analyze diet profiles of Eurasian otters inhabiting a marshy estuary area in Korea. We investigated their diet profiles based on spraint sampling followed by DNA metabarcoding analyses targeting 12S rRNA gene region for vertebrates, 16S rRNA gene region for invertebrates, and cytochrome c oxidase 1 (COI) gene region for fishes. For the vertebrate analysis, a blocking oligonucleotide (OBS1) was designed to suppress amplification of DNA fragments derived from the otters. The 12S rRNA gene sequencing assay detected species belonging to fishes (95%) and amphibians (3.3%). Fishes detected by 12S rRNA gene sequencing included crucian carp (Carassius auratus), mullets (Mugil spp.), bluegill (Lepomis macrochirus), and northern snakehead (Channa argus), which were also detected by COI gene sequencing. Among invertebrates, mud flat crabs (Helicana spp.) and shrimps (Palaemon spp.) were abundant. The designed blocking oligonucleotide OBS1 effectively inhibited amplification of the otter’s DNA, with only up to 0.21% of vertebrate sequence reads assigned to the otter. This study demonstrated that HTS-based DNA metabarcoding methods were useful to provide in-depth information regarding diet profiles of the otters at our sampling site. By using HTS-based DNA metabarcoding approaches, future research will explore detailed taxonomies of their diets across locations and seasons.欧亚水獭（Lutra lutra）是濒危物种，其饮食分析是保护工作的必要组成环节。该物种以鱼类等脊椎动物以及甲壳类等无脊椎动物为食，但由于传统形态学鉴定方法的分辨能力有限，其饮食的详细分类学信息尚未完全明晰。本研究采用基于高通量测序（high-throughput sequencing, HTS）的DNA宏条形码（DNA metabarcoding）技术，对栖息于韩国某沼泽河口区域的欧亚水獭的饮食组成进行分析。本研究通过水獭粪便采样结合DNA宏条形码分析开展相关研究，以脊椎动物的12S rRNA基因区域、无脊椎动物的16S rRNA基因区域以及鱼类的细胞色素c氧化酶亚基1（cytochrome c oxidase 1, COI）基因区域为扩增靶标。针对脊椎动物的分析，本研究设计了阻断寡核苷酸（blocking oligonucleotide, OBS1）以抑制水獭自身DNA片段的扩增。12S rRNA基因测序检测到的物种中，鱼类占比95%、两栖类占比3.3%；检测到的鱼类包括鲫（Carassius auratus）、鲻属（Mugil spp.）、蓝鳃太阳鱼（Lepomis macrochirus）以及乌鳢（Channa argus），上述类群同样被COI基因测序检测到。无脊椎动物中，泥滩蟹属（Helicana spp.）和长臂虾属（Palaemon spp.）为优势类群。所设计的阻断寡核苷酸OBS1可有效抑制水獭自身DNA的扩增，仅0.21%的脊椎动物序列读长被注释为水獭来源。本研究证实，基于HTS的DNA宏条形码方法可有效获取该采样点水獭饮食组成的详细信息。未来可依托基于HTS的DNA宏条形码技术，探究不同区域与季节下水獭饮食的详细分类学特征。