Environmental DNA (eDNA) analysis is gaining prominence as a tool for species and biodiversity monitoring in aquatic environments. eDNA shed by organisms is captured in grab samples, concentrated by filtration, extracted, and analyzed using molecular methods. Conventional capture and filtration methods are limited because (1) filtration does not capture all extracellular DNA, (2) eDNA can degrade during sample transport and storage, (3) filters often clog in turbid waters, reducing the eDNA captured, and (4) grab samples are time sensitive due to pulse eDNA inputs. To address these limitations, this work designs and validates Passive Environmental DNA Samplers (PEDS). PEDS consist of an adsorbent-filled sachet that is suspended in water to collect eDNA over time. Both extracellular and cellular DNA are captured, and the extracellular DNA is protected from degradation. The eDNA captured over time may be more representative than a grab sample. Two adsorbents, Montmorillonite Clay (MC) and Granular Activated Carbon (GAC), are tested. In laboratory experiments, MC-PEDS adsorbed five times more extracellular DNA and desorbed up to four times more than GAC-PEDS (despite high levels of eDNA loss during desorption). In microcosm and field experiments, GAC-PEDS captured over an order of magnitude more eDNA than MC-PEDS. Field results further validated PEDS as an effective eDNA capture method compared to conventional methods.
Stock assessments are critical to inform decisions for sustainable fisheries management. Environmental DNA (eDNA) analysis is a potential tool for assessing fish biomass and populations to aid in stock assessments. To facilitate modeling of biomass based on eDNA data, shedding and decay rates are needed. We designed species‐specific, probe‐based qPCR assays for three economically important fish species: black sea bass (Centropristis striata), winter flounder (Pseudopleuronectes americanus), and summer flounder (Paralichthys dentatus). Winter flounder eDNA was measured using two qPCR assays (135 and 292 bp). We report the eDNA shedding and decay rates and the associated variability from two replicate experimental systems. The eDNA decay rates were not significantly different between all species. The eDNA shedding rates between the two replicate systems were significantly different for winter flounder (135 bp assay) and summer flounder. qPCR amplicon length did not affect the eDNA decay rates for winter flounder. The three new qPCR assays were tested in environmental waters alongside traditional trawl surveys. No eDNA from BSB, WF, or SF was detected by eDNA methods, and out of 13 bottom trawls over 6 days only 1 WF, 1 SF, and 2 BSB were caught. This research presents three new, efficient qPCR assays and shows agreement between eDNA methods and trawl surveys suggesting low abundance or absence of target fish.
Bog turtles (Glyptemys muhlenbergii) are listed as Species of Greatest Conservation Need (SGCN) for wildlife action plans in every state it occurs and multi-state efforts are underway to better characterize extant populations and prioritize restoration efforts. However, traditional sampling methods can be ineffective due to the turtle’s wetland habitat, small size, and burrowing nature. Molecular methods, such as qPCR, provide the ability to overcome this challenge by effectively quantifying minute amounts of turtle DNA left behind in its environment (eDNA). Developing such methods for bog turtles has proved difficult partly because of the high sequence similarity between bog turtles and closely-related, cohabitating species, most often wood turtles (Glyptemys insculpta). Additionally, substrates containing bog turtle eDNA are often rich in organics or other substances that frequently inhibit both DNA extraction and qPCR amplification. Here, we describe the development and validation of a qPCR assay, BT3, targeting the mitochondrial cytochrome oxidase I gene that correctly identifies bog turtles with 100% specificity and sensitivity when tested on 201 blood samples collected from six species over a wide geographic range. We also developed a full-process internal control employing a genetically modified strain of Caenorhabditis elegans to improve DNA extraction methods, limit false negative results due to qPCR inhibition, and measure total DNA recovery from each sample. Using the internal control, we found that DNA recovery varied by over an order of magnitude between samples and likely explains the lack of bog turtle detection in some cases. Methods presented herein are highly-specific and may offer a more cost effective, non-invasive tool to supplement bog turtle population assessments in the Eastern United States. Poor or differential DNA recovery, which remains unmeasured in the vast majority of eDNA studies, significantly reduced the ability to detect bog turtle in their natural environment.
Environmental DNA (eDNA) once shed can exist in numerous states with varying behaviors including degradation rates and transport potential. In this study, we consider three states of eDNA: (1) a membrane-bound state referring to DNA enveloped in a cellular or organellar membrane, (2) a dissolved state defined as the extracellular DNA molecule in the environment without any interaction with other particles, and(3) an adsorbed state defined as extracellular DNA adsorbed to a particle surface in the environment. Capturing, isolating, and analyzing a target state of eDNA provides utility for better interpretation of eDNA degradation rates and transport potential.While methods for separating different states of DNA have been developed, they remain poorly evaluated due to the lack of state-controlled experimentation. We evaluated the methods for separating states of eDNA from a single sample by spiking DNA from three different species to represent the three states of eDNA as state-specific controls. We used chicken DNA to represent the dissolved state, cultured mouse cells for the membrane-bound state, and salmon DNA adsorbed to clay particles as the adsorbed state. We performed the separation in three water matrices, two environmental and one synthetic, spiked with the three eDNA states. The membrane-bound state was the only state that was isolated with minimal contamination from nontarget states. The membrane-bound state also had the highest recovery (54.11 ± 19.24%), followed by the adsorbed state (5.08 ± 2.28%), and the dissolved state had the lowest total recovery (2.21 ± 2.36%). This study highlights the potential to sort the states of eDNA from a single sample and independently analyze them for more informed biodiversity assessments. However, further method development is needed to improve recovery and reduce cross-contamination.
Environmental DNA (eDNA) analysis can be a powerful tool for the early detection of invasive organisms. However, research on terrestrial eDNA detection from foliage surfaces has been limited. In this study, we developed methods to capture and detect eDNA using qPCR from an invasive forest pest, hemlock woolly adelgid (Adelges tsugae), and three of its biological control predators Leucotaraxis piniperda, Leucotaraxis argenticollis, and Laricobius nigrinus. We designed four highly efficient qPCR assays with a low limit of detection (1-10 copies/reaction). The assay targeting A. tsugae was species-specific. The assays targeting Le. piniperda, and Le. argenticollis were biotype-specific in addition to being species-specific demonstrating applications of eDNA analysis beyond species-level detection. The La. nigrinus assay also detected DNA from closely related and hybridizing Laricobius rubidus. The eDNA methods were evaluated against traditional detection methods. We collected foliage samples from three strata (bottom, middle, and top) of eastern hemlock trees to detect the presence of A. tsugae. The detection of the biological control predators was evaluated using western hemlock foliage samples collected from the predators' native range in western Washington. The eDNA methods had significantly higher positive detection rates (2.8-4.5 times) than conventional methods of all target species. The strata of sampling were not significant in determining the presence of A. tsugae infestation.The eDNA concentration positively correlated with the observed density for all species. This study demonstrates the efficacy of eDNA analysis as a more sensitive tool for early detection of A. tsugae and to track the establishment of its biological control predators.
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