Using DNA metabarcoding and a novel canid-specific blocking oligonucleotide to investigate the composition of animal diets of raccoon dogs (Nyctereutes procyonoides) inhabiting the waterside area in Korea

Abstract

The raccoon dog (Nyctereutes procyonoides) is known to be an opportunistic generalist who feeds on a wide variety of foods. Historically, their diet has been investigated by morphological observation of undigested remains in feces, requiring specialized knowledge such as osteology, zoology, and phytology. Here, we used DNA metabarcoding of vertebrate 12S rRNA gene and invertebrate 16S rRNA gene to investigate their fecal contents. Additionally, we developed a blocking oligonucleotide that specifically inhibits the amplification of the canid 12S rRNA gene. We confirmed that the blocking oligonucleotide selectively inhibit the amplification of raccoon dog’s DNA without significantly changing the composition of the preys’ DNA. We found that the main foods of raccoon dogs in our study area, the waterside of paddy fields in Korea, were fishes such as Cyprinidae and insects such as mole crickets, which makes sense given the Korean fauna and their well-known opportunistic feeding behaviors. As a method to conveniently and objectively investigate feeding habits of raccoon dogs, this study provided baseline information on DNA metabarcoding. By using DNA metabarcoding, it is expected that the diet habits and ecology of raccoon dogs will be better understood by future research.

Combining vertebrate mitochondrial 12S rRNA gene sequencing and shotgun metagenomic sequencing to investigate the diet of the leopard cat (Prionailurus bengalensis) in Korea

“…The obiclean command was used to exclude erroneous reads. Using the ecotag command, the high-quality reads obtained were taxonomically assigned against our custom reference database [ 28 ] consisting of vertebrate mitochondrial 12S rRNA gene sequences downloaded from the EMBL nucleotide database. The database contains reference mitochondrial 12S rRNA gene sequences of a total of 12,574 vertebrate species.…”

“…The recent introduction of DNA barcoding in wildlife dietary analysis [ 23 – 28 ] has made it possible to overcome the difficulties described above. In carnivore and omnivore dietary studies [ 23 – 28 ], DNA markers such as the vertebrate mitochondrial 12S rRNA gene [ 29 ] have been targeted to analyze DNA derived from diets in collected fecal samples. DNA barcoding has also been applied to dietary analysis of leopard cats in Pakistan [ 30 ], China [ 31 , 32 ], and Korea [ 33 ].…”

Diversity of fecal parasitomes of wild carnivores inhabiting Korea, including zoonotic parasites and parasites of their prey animals, as revealed by 18S rRNA gene sequencing

“…Fecal samples previously collected for diet studies of the wild animals ( Kumari et al, 2019 ; Woo et al, 2022 , 2023 ) were used. A total of 40 fecal samples were collected, consisting of 11, 22, and 7 samples of raccoon dogs, leopard cats, and Eurasian otters, respectively.…”

“…A total of 40 fecal samples were collected, consisting of 11, 22, and 7 samples of raccoon dogs, leopard cats, and Eurasian otters, respectively. Sample metadata are listed in Supplementary Table 1 and sample collection methods are reported in our previous studies ( Kumari et al, 2019 ; Woo et al, 2022 , 2023 ). Briefly, fecal samples of raccoon dogs and Eurasian otters were collected from a waterside area ( Woo et al, 2022 ) and an estuary area ( Kumari et al, 2019 ), respectively.…”

“…Sample metadata are listed in Supplementary Table 1 and sample collection methods are reported in our previous studies ( Kumari et al, 2019 ; Woo et al, 2022 , 2023 ). Briefly, fecal samples of raccoon dogs and Eurasian otters were collected from a waterside area ( Woo et al, 2022 ) and an estuary area ( Kumari et al, 2019 ), respectively. Fecal samples of leopard cats were collected in inland areas ( Woo et al, 2023 ).…”