WWW.WAS.ORG • WORLD AQUACULTURE • DECEMBER 2023 33 (CONTINUED ON PAGE 34) Uncertainty: eDNA origination, unpredictable degradation over time, abiotic and biotic transport, and stochastic natural events Current population biology and ecology literature verifies numerous uncertainties with using, or relying on, sampling of DNA fragments. The uncertainties (e.g., origination, variable degradation over time, abiotic and biotic transport, stochastic natural events) have been discussed in the ecological literature, more so than the scientific literature focused on eDNA to detect aquatic invasive species (Harrison et al. 2019; Stewart 2019; Jerde 2021; Jo and Minamoto 2020; Wang et al. 2021; Joseph et al. 2022). Stewart (2019) provides an excellent analysis and Loeza-Quintana et al. (2020) and 11 associated papers for their argument supporting the need for improved eDNA validation, methods, and standardization, and point specifically to Harrison et al. (2019) for their incisive thinking. They write: “…uncertainties persist surrounding the physical processes that influence eDNA persistence and its fate within the environment. Because these techniques use fragments of DNA recovered from environmental samples to infer species presence, uncertainties in the relationship between the source organism(s) and the physical DNA molecules in the environment can significantly limit inferences made from eDNA-based tools and preclude their widespread application.” Harrison et al. (2019) also provided five notable recommendations to reduce errors that generate uncertainty: 1) integrate hydrological modelling into eDNA sampling; 2) increase use of replicated, controlled experiments in naturalized systems when studying processes that affect eDNA and estimates of uncertainty, designed with an understanding of the potential mechanisms that impact these processes; 3) eDNA parametrization and conclusions drawn from eDNA studies should be considered as ecosystem-specific given the significant differences in transport and attenuation mechanisms between lentic, lotic and marine ecosystems; 4) collect and include environmental data when collecting eDNA samples so that environmentally driven variation can eventually be assessed; and, 5) develop a full model predicting the relationships between eDNA and the organisms being studied to elucidate the relative contribution of individual decay and transport processes in environment-specific contexts that contribute to patterns of bias and noise in varying environments. Using modeling, Erickson et al. (2019) estimated samples sizes of a 3-level occurrence model (occurrence, capture and detection) to suggest, “detecting eDNA in ≥1 sample at a site required ≤ 15 samples per site for common species…detecting eDNA when looking for rare species required 45 to 90 samples per site.” Cristescu and Hebert (2018) described bioinformatics and taxonomic assignment challenges. Key to bioinformatics is designing primers to encompass the potential species encompassed by nationwide reporting. Relative to taxonomic identity, the authors noted, “Incomplete reference libraries and the presence of sequences derived from misidentified specimens mean that the species origin of many eDNA records remains uncertain” and “…users must ensure that reference databases are up-to-date and contain entries for species of interest. An accurate taxonomic assignment provides a robust way of linking genotype to phenotype…” Recent work by Danziger and Frederich (2022) emphasizes the critical importance of primer specificity. They were focused on developing appropriate primers for the European green crab (Carcinus maenas) in the Pacific Northwest. They found speciesspecific eDNA primers for species distributed world-wide, may need to be tested carefully against related local species. In this instance primers developed for C. maenas found in Maine led to gene amplification, not only of Pacific Northwest C. maenas, but also the Asian shore crab, Hemigrapsus sanguineus, the Rock crab, Cancer borealis, and the Jonah crab, Cancer irroratus. A concise examination by Lacoursière-Roussel and Deiner (2019) argued an integrated, multidisciplinary approach (i.e., life and physical sciences) is needed to create fundamental knowledge of what eDNA is and how it interacts with its surroundings. Until multidisciplinary analysis is accomplished, they noted, an accurate inference that a species was present in a place and time remains a challenge. As one of their several supporting examples, they reported: “…DNA in the environment has a fast degrading portion that is correlated with a species abundance, a portion that can remain detectable for weeks to months in water when the species is no longer present and a portion that can remain detectable for centuries in certain types of substrate such as lake sediments and permafrost.” Cristescu and Hebert (2018) spoke to the interaction of eDNA with the aquatic environment. Specific to one-off sampling for nonnative species, their comments reporting eDNA persistence in sediments is particularly problematical. They noted: “…eDNA in sediments can persist far longer and is often present at much higher concentration than is eDNA in the water column.” “…eDNA extracts from river sediments generated sequences of resident freshwater species, marine and estuarine species unlikely to occur at the sampled site, and freshwater species unrecorded for more than a century.” “Because aDNA [ancient DNA isolated from old specimens] from sediments may be resuspended, particularly in rapidly flowing rivers, DNA extracted from water may often contain eDNA that reflects historical deposits. Separating recent eDNA from aDNA is not straightforward. Moreover, discriminating between eDNA (particularly its cellular form) and genomic DNA from small organisms inadvertently captured during sampling is difficult.” Empirical research in lotic systems indicates fish eDNA can be detected 50 km (Laporte et al. 2020) to 130 km (Pont et al. 2018) from sources or 9 km from sources for crustaceans (Deiner and Altermatt 2014). The potential long-distance transport of eDNA by birds, vessels and flowing waters and its persistence in sediments creates, through false positive inference, significant species location, eradication or control challenges. The evolving diversity of farmed aquatic species over time at any particular farm will deposit eDNA in sediments that will be re-suspended during typical farm operations (e.g., seine harvest) or storm events. Similarly, eDNA entrained in lotic waters near farms or the eDNA persisting in sediments in those flowing waters may be sampled.
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