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

Abstract

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.

DNA-based detection of <i>Leptospira wolffii</i>, <i>Giardia intestinalis</i> and <i>Toxoplasma gondii</i> in environmental feces of wild animals in Korea

“…The fecal samples of Eurasian otters (n=7), leopard cats (n=22) and raccoon dogs (n=15) were collected from different sites in Korea. The otters’ spraints were previously collected in an estuary area in Ansan-si in Gyeonggi-do, with a centroid position at 37°16’22.6”N and 126°50’24.7”E, for their diet analyses on June 2017 [ 19 ]. The scat samples of leopard cats were collected in the Gongju-si and Yeongi-gun area in Chungcheongnam-do (n=14) at a centroid position at 36°32’2.86”N and 127°10’13.25”E, the Sangju-si area in Gyeongsangbuk-do (n=1) at 36°17’55.8”N and 128°7’27.12”E, and the Dalseong-gun and Goryeong-gun area in Daegu-si or Gyeongsangbuk-do (n=7) at a centroid position at 35°44’0.94”N and 128°24’10.24”E during February 2019.…”

The diet of otters (Lutra lutra) on the Agri river system, one of the most important presence sites in Italy: a molecular approach

“…DNA metabarcoding coupled with HTS represented a good approach for analyzing the diet of otters from Southern Italy using DNA extracted from non-invasive samples, in terms of accuracy, cost, time, and effort ( Deagle, Kirkwood & Jarman, 2009 ; Soininen et al, 2009 ; Pompanon et al, 2012 ; Berry et al, 2015 ; Kumari et al, 2019 ; Martínez-Abraín et al, 2020 ).…”

“…However, in the interpretation of data, we proceeded with caution. In fact, the use of reads count as a direct measure of ingested biomass remains a highly debated issue ( Deagle et al, 2019 ; Kumari et al, 2019 ) and some considerations should be taken into account before doing so. Ideally, the number of sequences assigned to a taxon would mirror the biomass in faecal material.…”

Peer Review #1 of "The diet of otters (Lutra lutra) on the Agri river system, one of the most important presence sites in Italy: a molecular approach (v0.1)"

“…DNA metabarcoding coupled with HTS represented a good approach for analyzing the diet of otters from Southern Italy using DNA extracted from non-invasive samples, in terms of accuracy, cost, time, and effort (Deagle, Kirkwood & Jarman, 2009;Soininen et al, 2009;Pompanon et al, 2012;Berry et al, 2015;Kumari et al, 2019;Martinez-Abrain et al, 2020).…”

“…However, in the interpretation of data, we proceeded with caution. In fact, the use of reads count as a direct measure of ingested biomass remains a highly debated issue (Deagle et al, 2019;Kumari et al, 2019) and some considerations should be taken into account before doing so.…”