Peer Review #2 of "Aquatic insect community structure revealed by eDNA metabarcoding derives indices for environmental assessment (v0.2)"

Abstract

Environmental DNA (eDNA) analysis provides an efficient and objective approach for monitoring and assessing ecological status; however, studies on the eDNA of aquatic insects, such as Ephemeroptera, Plecoptera, and Trichoptera (EPT), are limited despite its potential as a useful indicator of river health. Here, we investigated the community structures of aquatic insects using eDNA and evaluated the applicability of eDNA data for calculating assessment indices. Field surveys were conducted to sample river water for eDNA at six locations from upstream to downstream of two rivers in Japan in July and November 2016. Simultaneously, aquatic insects were collected using the traditional Surber net survey method. The communities of aquatic insects were revealed using eDNA by targeting the cytochrome oxidase subunit I gene in mitochondrial DNA via metabarcoding analyses. The eDNA results revealed 63 families and 75 genera of aquatic insects, which was double than that detected by the Surber net survey (especially for families in Diptera and Hemiptera). The seasonal differences of communities were discriminated by the eDNA and Surber net survey data. Furthermore, environmental assessment indices (i.e., EPT index and Chironomidae index) calculated using the richness of operational taxonomic units at the genus level showed positive correlations with total nitrogen concentration as a surrogate parameter of the degree of organic pollution but the index calculated using the Surber net survey data did not. Our results demonstrated that eDNA analysis with higher taxonomic resolution can serve as a more sensitive environmental assessment index than the traditional method that requires biotic samples.