Abstract Environmental DNA (eDNA) is a groundbreaking molecular tool that despite its challenges, offers a powerful and comprehensive method for surveying biodiversity. Insects, as key bioindicators of water quality in river ecosystems, make insect eDNA an especially promising tool for biomonitoring and water management. However, significant issues remain. This review explores historical and ongoing eDNA studies, focusing on the genetic diversity of insects, which underpins eDNA analysis. We address two key issues regarding eDNA metabarcoding: selecting suitable DNA barcoding regions for accurate molecular identification and the importance of DNA databases. A review of 82 papers showed that while the mitochondrial DNA (mtDNA) COI region is widely used, it often amplifies nontarget groups. Regarding primer concordance rates among insects, the mtDNA COI region, mixed nucleotide bases are often used to increase the match rate for insects. In contrast, primers targeting the mtDNA 16S rRNA region actually show higher match rates for insects. In addition, we highlight the critical role of regional DNA databases, particularly for species that exhibit genetic differentiation across regions. Also, using a local database, we demonstrated improved detection sensitivity, underscoring the importance of local databases for accurate species identification. To guide future database construction, we reviewed phylogeographic studies and using the Japanese Archipelago, which is a region with high species diversity, proposed genetic ecoregions that demonstrate genetic regionality globally. Our findings emphasize that insect eDNA is at the forefront of species identification technology, offering immense potential for future applications. With innovative approaches, it could revolutionize biomonitoring tools for diverse ecosystems.

ABSTRACT Environmental DNA (eDNA) is a powerful tool for biological monitoring that may overcome limitations of conventional macroinvertebrate surveys in running waters. However, the ability of eDNA to detect macroinvertebrate community changes immediately downstream of a perturbation, particularly in upstream–downstream comparisons, has not been adequately explored. To address this, we compared eDNA‐based assessments with macroinvertebrate surveys in a river receiving inflow from a metal‐contaminated tributary. Results from both eDNA‐based assessment and benthic macroinvertebrate collection revealed distinctly lower richness of taxa and zero‐radius operational taxonomic units (ZOTUs) at the metal‐contaminated tributary site compared to other study sites. Results from the collection of macroinvertebrates indicated that most richness and abundance metrics were significantly reduced at three metal‐contaminated sites located 150–1350 m downstream from the inflow of the tributary, compared with an upstream reference site. In contrast, the eDNA‐based assessment revealed similar ZOTU richness at the reference site and the three contaminated sites. Although statistically not significant because sample sizes were small, eDNA‐based nonmetric multidimensional scaling (NMDS) revealed some separation between the reference site and two downstream sites. However, no separation was apparent between the reference site and the site immediately downstream. This result suggested that eDNA at a site 150 m downstream from the inflow was likely affected by downstream drift of eDNA from the upstream reference area. That drift complicated the assessment of the community a short distance from the perturbation. The site separation detected by eDNA‐based assessment was promising, but the ZOTUs that contributed to the separation were mainly from dipteran taxa rather than from metal‐sensitive mayflies, which were significantly lower in abundance at the downstream, contaminated sites. Developing reliable local DNA barcoding information, particularly for these mayflies, may help overcome the limitations of making evaluations over relatively small spatial scales, such as upstream–downstream comparisons.