Abstract

Polyphylla laticollis manchurica (PLM), distributed across four regions along the Geumgang River in South Korea, is classified as endangered. Its developmental cycle from larva to adult spans 3-4 years, making it challenging to verify its presence or absence within its habitat, except during the adult phase. Over the last two decades, multiple noninvasive genomic DNA (gDNA) sampling methods, including ones using eggshells, feces, mucus, and molted skin, have been developed. These methods facilitate the identification of species and genetic analysis without causing harm to the target species. This study considers the highly conserved cytochrome b sequence region of PLM for PCR amplification using a direct gDNA extraction method with feces. To establish an effective direct gDNA extraction method, this study used DAP buffer – previously proven effective for extracting gDNA from feces and mucus of invertebrates – and compared PCR amplification efficiency based on feces weight and dilution factor. Optimal PCR amplification efficiency was achieved using 10 mg of feces per 100 μL DAP buffer and a 200-fold dilution. Species-specific PLM markers distinguish PLM from sympatric species within their habitats. The noninvasive species identification method employing feces permits rapid species identification by circumventing the sequencing process. This rapid identification technique could serve as a preliminary monitoring tool for the protection of the endangered PLM’s habitat when adult individuals are not visible.