32 DECEMBER 2023 • WORLD AQUACULTURE • WWW.WAS.ORG of five laboratories that participated in a highly prescribed blind proficiency testing study recorded false findings, albeit rarely (Sepulveda et al. 2020): “Rare instances of zebra or quagga mussel DNA amplification did occur in water bodies where one of the dreissenid mussel species is not known to occur, though only samples analyzed by Laboratory 4 amplified.” This amplifies concerns regarding the potential of false positives generated from samples collected and analyzed under less rigorous conditions. Among other sources, Farrell et al. (2021) describes benefits and uses of eDNA, and the potential for false positives: “Conversely, partly as a result of eDNA-based approaches being less likely to produce false negatives, they can be more prone to producing false positives (in comparison with eRNA-based studies and traditional studies) because of increased efficacy (detection of eDNA that does not come directly from a present or alive target species or pathogen…).” The science of eDNA is evolving rapidly, and new findings provide intriguing results which have implications for the validity of potential regulatory applications. For example, recent research has documented that eDNA can be airborne (Stokstad 2021; Clare et al. 2021; Clare et al. 2022), and DNA is found in bio-aerosols in the air (Mainelis 2020; Gusareva et al. 2022) including eDNA for aquatic animals. Four species of fish fed to zoo animals were detected in the air (Lynggaard et al. 2022), as was the eDNA of many different marine fish species at a dockside sampling site (Klepke et al. 2022). Airborne eDNA from different species held in separate holding tanks, ponds or raceways on a farm will intermingle. As an example, farms producing baitfish, sportfish, grass carp and other fish species, hold live fish before transport under open or closed sheds to protect them from weather, predators, or theft. Fish are separated by species into different vats. A shed may contain a number of different species, one or more of which may not be legal for sale in other states. The water in each vat receives constant vigorous aeration from a lowpressure blower or surface aerator. The airborne eDNA, as a bioaerosol, will circulate throughout the shed and adhere to other vats, dipnets, and even hauling tanks which are driven up close to or under the shed for loading. Living organisms constantly shed whole or fragmented deoxyribonucleic acid (DNA), in waste and reproductive products, in mucus, by touch, and even through the air. This ‘loose’ DNA is called environmental DNA (eDNA). Sampling aquatic environments for eDNA has gained considerable traction and focus since Ficetola et al. (2008) described their eDNA sampling of wetlands located in France to detect the nonnative American bullfrog (Rana catesbeiana). As the costs of DNA analysis have decreased, scientists have increasingly used it for species identification, biomonitoring, disease pathogen detection (e.g., Bass et al. 2023), and for identifying whole community assemblages. Many publications by research scientists now call for resource managers to embrace and adopt eDNA tools to supplement or replace traditional monitoring methods. There is no doubt that eDNA is a phenomenal advancement in science with incredible potential. At the same time, the global aquaculture community should take a hard look at eDNA and fully understand the benefits and limitations, as there are considerable uncertainties associated with eDNA sampling and interpretation. Regulatory Use of eDNA It has long been obvious that eDNA will eventually be used for regulation. The question is, what safeguards will be in place to ensure that results of eDNA testing are accurate? Before eDNA is used for testing in commercial aquaculture (either for monitoring or for regulatory purposes), every sampling protocol and test assay must be validated and standardized, participating laboratories must be nationally accredited, and each lab must participate in proficiency testing. This is no different than for other diagnostic tests. Numerous uncertainties have been documented in regards to eDNA, concerns which are exacerbated regarding its potential use for regulatory purposes, where false positives have the potential of causing significant harm. This concern is not unwarranted, given that natural resource conservation management often defaults to regulatory enforcement and litigation (Nie 2008). Although scientists involved in eDNA research are understandably enthusiastic about the repeatability and reproducibility of eDNA detections, one out A Hard Look at Environmental DNA Paul Zajicek and Nathan Stone FIGURE 1. eDNA sampling. Courtesy Fingerlakes Partnership for Regional Invasive Species Management.
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