Definitive environmental DNA research on aquatic insects: Analysis optimization using the recently developed MtInsects-16S primers set

Long-term biodiversity monitoring is necessary for the conservation and management of water resources. Notably, aquatic insects have been used as an indicator of water quality because they provide important basic information about freshwater ecosystems and water resources. Although environmental DNA (eDNA) surveys can enable easy and effective biomonitoring of aquatic insects, previous studies have not successfully detected all insect species, and there has been frequent amplification of nontarget taxa (e.g., algae and diatoms). Therefore, we developed a universal primers set, MtInsects-16S, for eDNA analyses of insects in the mtDNA 16SrRNA region. Furthermore, a well-established database of aquatic insects, especially the MtInsects-16S DNA region of Ephemeroptera, Plecoptera, and Trichoptera in Kanagawa Prefecture, which was the target area in this study, was constructed. Therefore, in this study, we conducted eDNA analyses using a universal primers set and using a well-established database. We conducted and compared capture surveys at the same sites to examine the detection capability of eDNA for Insecta. As a result, eDNA analyses using MtInsects- 16S not only detected almost all of the captured species but also detected many more species without amplifying nontarget taxa. This study demonstrated the application of eDNA analyses with unprecedented accuracy and reliability. It was also shown that community structure by eDNA reflected a relatively narrow range at the water sampling point. Although the data accumulation for constructing locally specific databases is an urgent issue, using the MtInsects-16S region is expected to be a breakthrough in the metabarcoding of insects.