Martina Lüthi

With accelerating biodiversity erosion, it is critical for species conservation to use rapid and scalable monitoring methods. One powerful biodiversity monitoring method that has emerged recently is environmental DNA (eDNA). eDNA analyses currently require lengthy protocols in well-equipped laboratories, slowing down the analyses and limiting applications across broad scales. Here, we developed a protocol for eDNA analyses, leveraging CRISPR-based diagnostic systems (Dx) with lateral flow tests to rapidly process and analyze eDNA samples on site. To test the versatility of the field-based protocol, we designed a CRISPR-Dx assay specific to the threatened and elusive African manatee (Trichechus senegalensis). We sampled water across ten locations in a national park in the Republic of Congo and detected manatee DNA directly on site in almost half of the sites. We later confirmed these detections with a high-sensitivity protocol in a well-equipped laboratory. The CRISPR-Dx detections were mainly confirmed with a previously reported quantitative PCR (qPCR) assay. Despite the lower sensitivity of the field-based protocol, our analysis shows that its speed and ease of application provide advantages over slower and more expensive methods to detect threatened and elusive species, in particular if the protocol is improved in the future. Our methodology will increase the accessibility to and speed of eDNA analyses, enhancing biodiversity monitoring efforts and species conservation initiatives.

While acquiring age information is crucial for efficient stock management and biodiversity conservation, traditional aging methods fail to offer a universal, non-invasive, and precise way of estimating a wild animal's age. DNA methylation from tissue DNA (tDNA) was recently proposed as a method to overcome these issues and showed more accurate results than telomere-length-based age assessments. Here, we used environmental DNA (eDNA) for the first time as a template for age estimation, focusing on the larval phase (10–24 days post-hatch) of cultured Dicentrarchus labrax (seabass), a species of major economic and conservation interest. Using third-generation sequencing, we were able to directly detect various modification types (e.g., cytosine and adenosine methylation in all contexts) across the whole genome using amplification-free nanopore sequencing. However, aging sites were only present in the mitogenome, which could be a specific feature of eDNA methylation or the consequence of better DNA protection within mitochondria. By considering qualitative and quantitative information about aging sites according to an objective model selection framework, our epigenetic clock reached a cross-validated accuracy of 2.6 days (Median Absolute Error). Such performances are higher than those of previous clocks, notably for adult seabass even when scaling MAE to the age range, which could be linked to a more dynamic epigenome during early life stages. Overall, our pilot study proposes new methods to determine the potential of eDNA for simultaneous age and biodiversity assessments, although robust validation of our preliminary results along with methodological developments are needed before field applications can be envisaged.

More efficient methods for extensive biodiversity monitoring are required to support rapid measures to address the biodiversity crisis. While environmental DNA (eDNA) metabarcoding and quantitative PCR (qPCR) methods offer advantages over traditional monitoring approaches, their large-scale application is limited by the time and labour required for developing assays and/or for analysis. CRISPR (clustered regularly interspaced short palindromic repeats) diagnostic technologies (Dx) may overcome some of these limitations, but they have been used solely with species-specific primers, restricting their versatility for biodiversity monitoring. Here, we demonstrate the feasibility of designing species-specific CRISPR-Dx assays in silico within a short metabarcoding fragment using a general primer set, a methodology we term ‘ampliscanning’, for 18 of the 22 amphibian species in Switzerland. We sub-selected nine species, including three classified as regionally endangered, to test the methodology using eDNA sampled from ponds at nine sites. We compared the ampliscanning detections to data from traditional monitoring at these sites. Ampliscanning was successful at detecting target species with different prevalences across the landscape. With only one visit, we detected more species per site than three traditional monitoring visits (visual and acoustic detections by trained experts), in particular more elusive species and previously undocumented but expected populations. Ampliscanning detected 25 species/site combinations compared to 12 with traditional monitoring. Sensitivity analyses showed that larger numbers of field visits and PCR replicates are more important for reliable detection than many technical replicates at the CRISPR-Dx assay level. Given the reduced sampling and analysis effort, our results highlight the benefits of eDNA and CRISPR-Dx combined with universal primers for large-scale monitoring of multiple endangered species across landscapes to inform conservation measures.