An Optimized Environmental DNA Method to Improve Detectability of the Endangered Sichuan Taimen (Hucho bleekeri)

Abstract

Environmental DNA (eDNA) techniques have emerged as a cost-effective and non-invasive strategy for monitoring the distribution of endangered aquatic species. Despite their numerous advantages, operational uncertainty at each step of the process represents a significant challenge that can impact the reliability of results. Hence, the optimization of the eDNA analytic protocol is of utmost importance. In this study, the rare and endangered fish species Sichuan taimen was chosen as the target species to evaluate the concordance between different approaches (tissue, pond, and field water samples) for eDNA detection. The results showed that membrane filtration, collection of a 2-L water sample, and utilization of the DNeasy Tissue and Blood DNA extraction kit were suitable for the analysis of DNA in water samples. Furthermore, the mtDNA D-loop region demonstrated potential as a specific molecular marker for the precise identification of Sichuan taimen. Our results indicated that TaqMan fluorescence PCR can serve as the optimal detection method for reliable identification of target DNA even at extremely low concentrations in the field. This study established an optimized eDNA analysis protocol for Sichuan taimen detection, which will provide the basis for future resource investigations aimed at protection of this endangered aquatic species.

Environmental DNA Detects Remaining Populations of Endangered Stream Salmon (Sichuan Taimen: Hucho bleekeri Kimura Salmonidae) in the Qinling Mountains

“…In 2018, researchers using underwater visual observations and gill netting discovered the presence of wild young-of-the-year taimens in the river. The Xushui River is located on the northern latitude 33 In the first phase of this study, we followed a protocol for detecting targeted DNA of Sichuan taimen in water collected from culture ponds [25]. A 236 bp D-loop region of mtDNA and common PCR were employed to compare the eDNA detection rates across different seasons to determine the best time for the distribution survey of Sichuan taimen.…”

“…In seasonal effect analysis, although the common PCR amplification method can obtain bright and single-target bands for the detection of culture water samples, occasionally there were several non-specific bands in the electrophoretic image when eDNA was amplified from the stream water samples. Therefore, a more sensitive TaqMan qPCR amplification procedure described by Deng [25] was applied to detect the targeted eDNA of Sichuan taimen along the Taibai River and the Xushui River.…”

“…Detailed geographic information for all sampling sites can be found in Table 1, and the corresponding location map is provided in Figure 2. However, to ensure species protection, the geographic information for an additional three Water eDNA was extracted and amplified using a species-specific primer employing the same lab procedure described by Deng [25]. PCR products were run on 1% agarose gels to determine success, then purified using a Qiagen Min Elute PCR purification kit (Qiagen GmbH, Hilden, Germany), and were commercially sequenced in both forward and reverse directions on ABI3730 (Applied Biosystems, Foster City, CA USA).…”