Environmental DNA (EDNA) is DNA shed by living organisms into the environment through sloughed skin or scales, mucus, feces, or urine

This Cooperative Agreement award, provided by the U.S. Department of the Interior's Fish and Wildlife Service, supports the next phase of developing environmental DNA (eDNA) based monitoring approaches for non-invasive monitoring of California vernal pool ecosystems under the National Wildlife Refuge System Enhancements program (CFDA 15.654).

The $144,004 award will enable the University of California, Davis to finalize statistical analyses of eDNA metabarcoding data collected in prior phases, design a rapid SHERLOCK assay for detecting the vernal pool fairy shrimp (Branchinecta lynchi), and begin assay development for the California tiger salamander (Ambystoma californiense). The assays will be field tested at the Stone Lakes National Wildlife Refuge and Don Edwards San Francisco Bay National Wildlife Refuge. This work will advance the use of innovative eDNA techniques to provide community-level biodiversity inventories and enable rapid, non-invasive monitoring of target species within these sensitive vernal pool ecosystems.

Quantitative PCR (QPCR), high throughput sequencing, or crispr based techniques can detect EDNA in environmental samples, allowing biologists to determine the presence or absence of a species at a sampling location without having to directly observe or capture organisms. EDNA detection exploits DNA barcodes, or gene regions that vary between taxa, and can target single species or multiple species. Single species detection, which is useful when only one or a small number of species are targeted, involves amplification of a species-specific sequence on a QPCR machine or a handheld fluorescence reader. Multi-species detection, also known as EDNA metabarcoding, uses universal primers to amplify one or more barcoding regions for all DNA in a sample. These amplified regions then undergo high throughput sequencing and are sorted bioinformatically. EDNA metabarcoding can provide community-level inventories of sampling locations (LIM ET AL. 2016 port ET AL. 2016) which can be useful for managing ecosystems where biodiversity is poorly characterized. Under this cooperative agreement we propose to complete the fourth phase in developing EDNA based approaches for non-invasive monitoring of california vernal pool ecosystems. We will finalize our statistical analyses of EDNA metabarcoding data collected in phases I-III. We will also finalize design of the sherlock assay for vernal pool fairy shrimp (brachinecta lynchi) and begin assay design for california tiger salamander (ambystoma californiense). Assays for vernal pool fairy shrimp and california tiger salamander will be field tested at stone lakes national wildlife refuge (NWR) and DON edwards SAN francisco BAY NWR, respectively, in this phase. Sherlock (specific high-sensitivity enzymatic reporter unlocking) assays will allow for rapid (30 mins or less baerwald ET AL. 2020, 2023) detection of target species from an EDNA sample taken in the field without the need for molecular expertise or expensive instrumentation. Below we describe how we plan to accomplish this fourth phase.