Quantitative PCR assays to detect whales, rockfish, and common murre environmental DNA in marine water samples of the Northeastern Pacific

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Andruszkiewicz, E. A., Yamahara, K. M., Closek, C. J., & Boehm, A. B. Quantitative PCR assays to detect whales, rockfish, and common murre environmental DNA in marine water samples of the Northeastern Pacific. Plos One, 15(12), (2020): e0242689, doi:10.1371/journal.pone.0242689.Monitoring aquatic species by identification of environmental DNA (eDNA) is becoming more common. To obtain quantitative eDNA datasets for individual species, organism-specific quantitative PCR (qPCR) assays are required. Here, we present detailed methodology of qPCR assay design and testing, including in silico, in vitro, and in vivo testing, and comment on the challenges associated with assay design and performance. We use the presented methodology to design assays for three important marine organisms common in the California Current Ecosystem (CCE): humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge). All three assays have excellent sensitivity and high efficiencies ranging from 92% to 99%. However, specificities of the assays varied from species-specific in the case of common murre, genus-specific for the shortbelly rockfish assay, and broadly whale-specific for the humpback whale assay, which cross-amplified with other two other whale species, including one in a different family. All assays detected their associated targets in complex environmental water samples.This work is a contribution to the Marine Biodiversity Observation Network (MBON). The MBON project was supported by NASA grant NNX14AP62A ‘National Marine Sanctuaries as Sentinel Sites for a Demonstration Marine Biodiversity Observation Network (MBON)’ funded under the National Ocean Partnership Program (NOPP RFP NOAA-NOS-IOOS-2014-2003803 in partnership between NOAA, BOEM, and NASA), and the U.S. Integrated Ocean Observing System (IOOS) Program Office

Modeling characterization of the vertical and temporal variability of environmental DNA in the mesopelagic ocean

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Allan, E. A., DiBenedetto, M. H., Lavery, A. C., Govindarajan, A. F., & Zhang, W. G. Modeling characterization of the vertical and temporal variability of environmental DNA in the mesopelagic ocean. Scientific Reports, 11(1), (2021): 21273, https://doi.org/10.1038/s41598-021-00288-5.Increasingly, researchers are using innovative methods to census marine life, including identification of environmental DNA (eDNA) left behind by organisms in the water column. However, little is understood about how eDNA is distributed in the ocean, given that organisms are mobile and that physical and biological processes can transport eDNA after release from a host. Particularly in the vast mesopelagic ocean where many species vertically migrate hundreds of meters diurnally, it is important to link the location at which eDNA was shed by a host organism to the location at which eDNA was collected in a water sample. Here, we present a one-dimensional mechanistic model to simulate the eDNA vertical distribution after its release and to compare the impact of key biological and physical parameters on the eDNA vertical and temporal distribution. The modeled vertical eDNA profiles allow us to quantify spatial and temporal variability in eDNA concentration and to identify the most important parameters to consider when interpreting eDNA signals. We find that the vertical displacement by advection, dispersion, and settling has limited influence on the eDNA distribution, and the depth at which eDNA is found is generally within tens of meters of the depth at which the eDNA was originally shed from the organism. Thus, using information about representative vertical migration patterns, eDNA concentration variability can be used to answer ecological questions about migrating organisms such as what depths species can be found in the daytime and nighttime and what percentage of individuals within a species diurnally migrate. These findings are critical both to advance the understanding of the vertical distribution of eDNA in the water column and to link eDNA detection to organism presence in the mesopelagic ocean as well as other aquatic environments.This research is part of the Woods Hole Oceanographic Institution’s Ocean Twilight Zone project, funded as part of The Audacious Project housed at TED

Adoption of environmental DNA in public agency practice

Abstract Environmental DNA (eDNA) analysis has matured to the point that it is ready for deployment in many applications, particularly in aquatic environments. But public agencies have yet to adopt eDNA methods into their environmental decision making routines at scale, even when eDNA offers clear advantages to those now in use. This article provides a perspective on this gap by considering adoption of a new technology as a path‐dependent, social process in which some paths lead to outcomes that provide far greater benefits than others. We use the diffusion of innovations to investigate eDNA adoption by government agencies, in an effort to (1) identify likely paths toward institutionalized uptake, and (2) suggest ways of avoiding societally inefficient adoption outcomes. Because—as in any scientific endeavor—the best eDNA practices will continue to change, public agencies must remain open to improvements while building a useful and validated array of methods for routine eDNA application. Here we suggest one mechanism for doing so, which we call collaborative governance

Critical considerations for communicating environmental DNA science

Abstract The economic and methodological efficiencies of environmental DNA (eDNA) based survey approaches provide an unprecedented opportunity to assess and monitor aquatic environments. However, instances of inadequate communication from the scientific community about confidence levels, knowledge gaps, reliability, and appropriate parameters of eDNA‐based methods have hindered their uptake in environmental monitoring programs and, in some cases, has created misperceptions or doubts in the management community. To help remedy this situation, scientists convened a session at the Second National Marine eDNA Workshop to discuss strategies for improving communications with managers. These include articulating the readiness of different eDNA applications, highlighting the strengths and limitations of eDNA tools for various applications or use cases, communicating uncertainties associated with specified uses transparently, and avoiding the exaggeration of exploratory and preliminary findings. Several key messages regarding implementation, limitations, and relationship to existing methods were prioritized. To be inclusive of the diverse managers, practitioners, and researchers, we and the other workshop participants propose the development of communication workflow plans, using RACI (Responsible, Accountable, Consulted, Informed) charts to clarify the roles of all pertinent individuals and parties and to minimize the chance for miscommunications. We also propose developing decision support tools such as Structured Decision‐Making (SDM) to help balance the benefits of eDNA sampling with the inherent uncertainty, and developing an eDNA readiness scale to articulate the technological readiness of eDNA approaches for specific applications. These strategies will increase clarity and consistency regarding our understanding of the utility of eDNA‐based methods, improve transparency, foster a common vision for confidently applying eDNA approaches, and enhance their benefit to the monitoring and assessment community

Signal and noise in metabarcoding data.

Metabarcoding is a powerful molecular tool for simultaneously surveying hundreds to thousands of species from a single sample, underpinning microbiome and environmental DNA (eDNA) methods. Deriving quantitative estimates of underlying biological communities from metabarcoding is critical for enhancing the utility of such approaches for health and conservation. Recent work has demonstrated that correcting for amplification biases in genetic metabarcoding data can yield quantitative estimates of template DNA concentrations. However, a major source of uncertainty in metabarcoding data stems from non-detections across technical PCR replicates where one replicate fails to detect a species observed in other replicates. Such non-detections are a special case of variability among technical replicates in metabarcoding data. While many sampling and amplification processes underlie observed variation in metabarcoding data, understanding the causes of non-detections is an important step in distinguishing signal from noise in metabarcoding studies. Here, we use both simulated and empirical data to 1) suggest how non-detections may arise in metabarcoding data, 2) outline steps to recognize uninformative data in practice, and 3) identify the conditions under which amplicon sequence data can reliably detect underlying biological signals. We show with both simulations and empirical data that, for a given species, the rate of non-detections among technical replicates is a function of both the template DNA concentration and species-specific amplification efficiency. Consequently, we conclude metabarcoding datasets are strongly affected by (1) deterministic amplification biases during PCR and (2) stochastic sampling of amplicons during sequencing-both of which we can model-but also by (3) stochastic sampling of rare molecules prior to PCR, which remains a frontier for quantitative metabarcoding. Our results highlight the importance of estimating species-specific amplification efficiencies and critically evaluating patterns of non-detection in metabarcoding datasets to better distinguish environmental signal from the noise inherent in molecular detections of rare targets

Toward a national eDNA strategy for the United States

Abstract Environmental DNA (eDNA) data make it possible to measure and monitor biodiversity at unprecedented resolution and scale. As use‐cases multiply and scientific consensus grows regarding the value of eDNA analysis, public agencies have an opportunity to decide how and where eDNA data fit into their mandates. Within the United States, many federal and state agencies are individually using eDNA data in various applications and developing relevant scientific expertise. A national strategy for eDNA implementation would capitalize on recent scientific developments, providing a common set of next‐generation tools for natural resource management and public health protection. Such a strategy would avoid patchwork and possibly inconsistent guidelines in different agencies, smoothing the way for efficient uptake of eDNA data in management. Because eDNA analysis is already in widespread use in both ocean and freshwater settings, we focus here on applications in these environments. However, we foresee the broad adoption of eDNA analysis to meet many resource management issues across the nation because the same tools have immediate terrestrial and aerial applications