Effects of sampling strategies and DNA extraction methods on eDNA metabarcoding: A case study of estuarine fish diversity monitoring.

Environmental DNA (eDNA) integrated with metabarcoding is a promising and powerful tool for species composition and biodiversity assessment in aquatic ecosystems and is increasingly applied to evaluate fish diversity. To date, however, no standardized eDNA-based protocol has been established to monitor fish diversity. In this study, we investigated and compared two filtration methods and three DNA extraction methods using three filtration water volumes to determine a suitable approach for eDNA-based fish diversity monitoring in the Pearl River Estuary (PRE), a highly anthropogenically disturbed estuarine ecosystem. Compared to filtration-based precipitation, direct filtration was a more suitable method for eDNA metabarcoding in the PRE. The combined use of DNeasy Blood and Tissue Kit (BT) and traditional phenol/chloroform (PC) extraction produced higher DNA yields, amplicon sequence variants (ASVs), and Shannon diversity indices, and generated more homogeneous and consistent community composition among replicates. Compared to the other combined protocols, the PC and BT methods obtained better species detection, higher fish diversity, and greater consistency for the filtration water volumes of 1 000 and 2 000 mL, respectively. All eDNA metabarcoding protocols were more sensitive than bottom trawling in the PRE fish surveys and combining two techniques yielded greater taxonomic diversity. Furthermore, combining traditional methods with eDNA analysis enhanced accuracy. These results indicate that methodological decisions related to eDNA metabarcoding should be made with caution for fish community monitoring in estuarine ecosystems.

Stochastic determination of the spatial variation of potentially pathogenic bacteria communities in a large subtropical river.

Understanding the composition and assembly mechanism of waterborne pathogen is essential for preventing the pathogenic infection and protecting the human health. Here, based on 16S rRNA sequencing, we investigated the composition and spatial variation of potentially pathogenic bacteria from different sections of the Pearl River, the most important source of water for human in Southern China. The results showed that the potential pathogen communities consisted of 6 phyla and 64 genera, covering 11 categories of potential pathogens mainly involving animal parasites or symbionts (AniP), human pathogens all (HumPA), and intracellular parasites (IntCelP). Proteobacteria (75.87%) and Chlamydiae (20.56%) were dominant at the phylum level, and Acinetobacter (35.01%) and Roseomonas (8.24%) were dominant at the genus level. Multivariate analysis showed that the potential pathogenic bacterial community was significantly different among the four sections in the Pearl River. Both physicochemical factors (e.g., NO3-N, and suspended solids) and land use (e.g., urban land and forest) significantly shaped the pathogen community structure. However, spatial effects contributed more to the variation of pathogen community based on variation partitioning and path analysis. Null model based normalized stochasticity ratio analysis further indicated that the stochastic process rather than deterministic process dominated the assembly mechanisms by controlling the spatial patterns of potential pathogens. In conclusion, high-throughput sequencing shows great potential for monitoring the potential pathogens, and provided more comprehensive information on the potentially pathogenic community. Our study highlighted the importance of considering the influences of dispersal-related processes in future risk assessments for the prevention and control of pathogenic bacteria.