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Affiliation: The eDNA Collaborative, University of Washington

Email: ednacollab@uw.edu

Author: The eDNA Collaborative


The analysis of environmental DNA (eDNA) is in its infancy, as hundreds of researchers around the world begin to develop a huge diversity of techniques and applications. Out of this ferment, we believe great ideas will quickly emerge and be widely adopted through bottom-up, grassroots exchanges among users. The faster and more routine those exchanges are, the more efficiently knowledge can spread.

The best available techniques will be most useful when they are transparent, repeatable, and freely available. Practitioners will then be more likely to gravitate toward a common set of tools. Where future regulatory and management applications demand standardization, the relevant standards will arise out of this common set of tools and practices.

  • Title: Can we count salmon with a low-tech eDNA sampling robot?

Affiliation: Smith-Root

Email: athomas@smith-root.com

Author: Austen Thomas

Abstract: Longitudinal eDNA sampling of remote or difficult to access environments can be costly due to the amount of human labor required to collect samples over time. Thus, there is need for systems capable of being deployed for weeks or months that can automatically collect eDNA samples at regular intervals without the need of a human sampler. Existing eDNA sampling robots are prohibitively expensive for many projects and their reliance on automated DNA extraction or liquid preservation methods requires complex, specialized componentry. The advent of self-preserving eDNA filter housings that desiccate the encapsulated membranes has created an opportunity to build a simplified eDNA auto-sampling system. In this study we test a prototype eDNA autosampler (Smith-Root) that is capable of autonomously collecting 16 self-preserving eDNA samples, and we intend to compare a time series of eDNA qPCR SQ values for two salmon species with their associated fish counts at a nearby fishway. In June 2022 the autosampler was installed on the dock at the King County Environmental Lab (WA), in the Fremont Cut, approximately 2.5 km upstream of the Ballard Locks where the migrations of Chinook (Oncorhynchus tshawytscha) and sockeye (Oncorhynchus nerka) salmon are monitored. The sampler is currently programmed to collect a 5L sample every 12h, and we conduct weekly servicing of the system to collect the archived samples and replace them with fresh filters. Do date, 122 samples have been collected by the autosampler, encompassing large portions of both the sockeye and Chinook migrations through the Lake Washington Ship Canal.

Affiliation: National Museum of Natural History, Smithsonian Institution

Email: StepienCA@si.edu

Author: Carol Stepien

Coauthors: Matthew E. Neilson2, Nathaniel T. Marshall3, Matthew R. Snyder4, and Julie E. Keister5

Coauthor affiliations:

2Matthew E. Neilson, U.S. Geological Survey, Nonindigenous Aquatic Species Database,

Wetland and Aquatic Research Center, Gainesville, FL 32653. mneilson@usgs.gov

3Nathaniel T. Marshall, Stantec Consulting Ltd., Columbus, Ohio 43204 nathaniel.marshall@stantec.com

4Matthew R. Snyder, Bioinformatics Consultant, Private Industry, Seattle, WA 98177 msnyder424@gmail.com

5Julie E. Keister, School of Oceanography, University of Washington, Seattle, WA 98195 keisterj@uw.edu

Abstract: Metabarcoding assays offer the opportunity to identify and track aquatic invasive species (AIS) from samples of environmental (e) DNA and bulk mixed organismal samples (e.g., plankton, sediment, diet contents). Assays may be targeted (taxon-specific) or general in scope and involve mitochondrial (mt) and/or nuclear (n) DNA markers, which identify taxon-diagnostic SNPs (single nucleotide polymorphisms). Metabarcoding allows researchers and managers to rapidly survey an entire community’s composition to identify both abundant species and rare and/or cryptic taxa (including newly introduced species) that may be overlooked by conventional sampling methods. Metabarcoding tools allow management agencies the opportunity to target and track invasive species, verified either with traditional sampling, or continued eDNA water and/or bulk organism capture. Additionally, more specialized DNA assays can be employed (e.g., quantitative (q) PCR, and/or targeted species-specific metabarcoding assays), which can detect and be useful for relatively quantifying a given species and its population variants.  Here we give several examples of recent findings about AIS and management actions/implications in marine, estuarine, and freshwater systems, as well as the increased use of metabarcoding in these efforts, over the last 5 years.

  • Title: Complementary eDNA and morphological surveys detect estuarine and coastal transport of invasive species in Lake Superior

Affiliation: University of Minnesota – Duluth and US EPA Great Lakes Toxicology and Ecology Division

Email: courtneyelarson126@gmail.com

Author: Courtney E Larson

Abstract: Surface currents are an important vector for biological invasions in marine systems and represent an understudied component of species spread in the Great Lakes. Through DNA, morphological, and environmental surveys in 2017 and 2019 along the south shore of Lake Superior, we demonstrated Dreissena (a group of two invasive mussels) veliger (planktonic early life stage) transport from an invaded estuary (Saint Louis River Estuary – SLRE) to a distant archipelago (Apostle Islands National Lakeshore) by nearshore currents. These currents and environmental conditions can be variable, and increasingly so with climate change; therefore, early detection monitoring for Dreissena along previously inhospitable environmental gradients is important for continued conservation of Lake Superior biodiversity and habitat. The 2019 survey found an unexpectedly high proportion of quagga mussel (D. bugensis) eDNA at the station located within the SLRE, where previous studies and the environmental conditions had led us to expect the vast majority of the Dreissena to be zebra mussels (D. polymorpha). A 2022 survey in the SLRE will investigate the species composition of the SLRE Dreissena, applying molecular techniques that enable species-level differentiation of veligers and juveniles. These studies demonstrate methods can iteratively build on each other over multiple surveys as technology develops, management concerns change, and research questions arise.

Affiliation: Johns Hopkins Applied Physics Laboratory

Email: hayley.dehart@jhuapl.edu

Author: Hayley M. DeHart

Coauthors: K. Zudock, P. Carey, and P. Thielen

Abstract: Marine mammals are comprised of at least 129 unique species across the world’s oceans. Protected under the US Marine Mammals Protection Act, they represent an ideal species group to monitor using eDNA approaches, both due to challenges in their detection and their ecological roles in the world’s oceans. Our group is working to make marine mammal eDNA characterization more efficient, comprehensive, and interpretable as a first step in adopting these technologies for monitoring purposes. To address these needs, we first identified gaps in mitochondrial genome characterization across all marine mammal species through in-depth analysis of species/subspecies designations and consolidations of publicly available mitochondrial genomes. We then used this custom marine mammal database to establish a field-capable bioinformatics approach for marine mammal eDNA classification. These approaches were utilized to establish permanent on-ship eDNA analysis capabilities in the Arctic, successfully recovering eDNA sequences from verified sightings of Walrus (Odobenus rosmarus), and an otherwise unidentifiable whale carcass that was characterized as a sperm whale (Physeter macrocephalus). These results demonstrate significant promise for advanced eDNA-based marine mammal research to study the presence, absence, and distribution of these protected species with potential to improve conservation practices.

Affiliation: Lehigh University

Email: ljm419@lehigh.edu

Author: Luke McCartin

Abstract: Environmental DNA (eDNA) sequencing and quantification complement established tools to assess the biodiversity of vulnerable benthic ecosystems. Offshore, marine eDNA may be transported long distances by complex ocean currents until it dilutes or degrades and is no longer detectable using PCR-based methods. A comprehensive understanding of the impact of marine physicochemical conditions on eDNA persistence has not been established. This gap in knowledge obscures the source location of eDNA from benthic ecosystems. In the mesophotic and deep ocean, below 30 meters depth, temperature, pH and dissolved oxygen concentration vary with depth and location. We conducted eDNA persistence experiments using eDNA derived from a deep-sea coral to determine the degradation rate among the natural combinations of temperature, pH and dissolved oxygen concentration that occur commonly in the deep ocean. The eDNA degradation process was best explained by a model with two phases with different decay rate constants. During the initial phase, eDNA degraded rapidly, and the rate was independent of physicochemical factors. During the second phase, eDNA degraded slowly, and the rate was strongly controlled by temperature and weakly controlled by pH. We did not find evidence that dissolved oxygen concentration from 0.1 mg/L to fully saturated conditions influenced eDNA degradation rate. Based on our results and publications using similar methods, we estimate that marine eDNA can persist at quantifiable concentrations for over 2 weeks at low temperatures (≤ 10 °C) but for a week or less at ≥ 20 °C. We propose a general temperature-dependent model to predict the maximum persistence time of eDNA detectable through single-species eDNA quantification methods. The relationship between temperature and eDNA persistence appears to be independent of the source taxa, however further experiments with other marine invertebrates are necessary. Community-wide sequencing data from eDNA samples at an offshore, mesophotic reef will test predictions regarding the transport extent of eDNA from benthic organisms and the application of eDNA analyses in these habitats. This research is supported by NOAA OER award NA18OAR0110289.

Affiliation: Stanford University

Email: mshea@stanford.edu

Author: Meghan Shea

Abstract: Interest is growing in using environmental DNA (eDNA) to study biodiversity in marine environments, which is increasingly important as humans alter ocean ecosystems faster than we can currently study them. Yet many knowledge gaps remain, both scientifically and sociopolitically, in how to deploy eDNA technologies at scale. My doctoral work involves studying the potential of eDNA using approaches and methods–from qualitative interviewing to laboratory science–across many disciplines, including science & technology studies, sociology, oceanography, and environmental engineering. Here, I present four in-progress projects that showcase a wide variety of academic approaches to studying the growing promise of eDNA.
1) In a systematic review of marine eDNA metabarcoding studies, I am analyzing how these studies store and contextualize their sequence data, to understand potential challenges to data access and re-use.
2) By collecting eDNA samples in individual tide pools at high frequency over the period that the intertidal ecosystem is exposed, I am analyzing the spatial and temporal scale of eDNA detection possible in complex, heterogeneous coastal environments.
3) In interviews with practitioners using eDNA within community science projects, as well as focus groups with non-experts collecting eDNA samples for the first time, I am characterizing the many values of projects designed to put eDNA tools in the hands of the public and the challenges faced when doing this work.
4) Using participant observations of an oceanographic cruise integrating eDNA sampling into its long-term monitoring program for the first time, as well as interviews with cruise scientists, I am studying what happens in practice when eDNA is brought into new contexts for both scientific and management aims.

  • Title: High-frequency and long-term observations of endangered salmonid eDNA in a coastal stream: temporal dynamics, relationships with environmental factors, and comparisons to organism abundance

Affiliation: Stanford University

Email: rtsearcy@stanford.edu

Author: Ryan Searcy

Abstract: Environmental DNA (eDNA) can be used to detect invasive species, identify the presence of biological pollutants, and characterize biodiversity, but a greater understanding of how eDNA behaves in the environment over multiple time scales is needed before eDNA can be a tool for ecosystem monitoring applications. The objectives of this study were to use autonomous methods to conduct long-term, high-frequency monitoring of native salmonid and invasive species eDNA in a Californian coastal stream; describe eDNA behavior by assessing temporal variation on multiple scales and identifying important environmental factors that drive this variation; and evaluate the utility of the eDNA datasets in detecting rare species and representing organismal abundance. Using environmental sample processors (ESPs) — high-throughput autonomous samplers — and qPCR, we enumerated eDNA concentrations from 674 water samples collected at sub-daily intervals over 360 days at a single site. eDNA from two threatened salmonid species (O. kisutch and O. mykiss) was detected in the majority of samples, but eDNA from two invasive species (M. saxatilis and P. antipodarum) was detected in less than 1% of samples below the level of quantification. O. kisutch eDNA was generally in lower concentration and more variable than O. mykiss eDNA. High-frequency (i.e. subdaily and daily) variability in salmonid eDNA concentrations showed occasional patchiness (i.e large differences between consecutive samples), while seasonal differences were observed to correspond to the natural history of the species at this site. Salmonid eDNA was significantly associated with creek discharge, photoperiod, and whether the creek mouth was open or bermed. We compared the salmonid eDNA signals to fish abundance data collected from weir traps located at the site. Fish were detected in the stream more often by eDNA sampling than from trapping. Significant, positive associations between fish abundance (i.e. number of individuals or biomass) and eDNA concentrations were observed for O. mykiss; however, no such associations were observed for O. kisutch. Our results inform future design of autonomous eDNA sampling studies.

  • Title: eDNA, Estuaries and End users – Building a standardized eDNA sampling program to support resource management in the National Estuarine Research Reserves Affiliation: University of New Hampshire

Email: jthmiller@icloud.com

Author: Jeffrey T. Miller

Coauthors: Jeffrey T. Miller, University of New Hampshire; Jason Garwood, Apalachicola FL; Christopher Peter, Great Bay NH; Nikki Dix, Guana Tolomato Matanzas FL; Shimi Rii, He’eia HI; Thomas Grothues, Jacques Cousteau NJ; Edward J. Buskey  Mission-Aransas, TX; Sylvia Yang  Padilla Bay, WA; Matt Ferner San Francisco Bay, CA; Shon Schooler, South Slough OR; Jason Goldstein, Wells ME and Alison Watts, University of New Hampshire.

Abstract: eDNA is a potentially powerful tool for estuarine assessment, but the application and interpretation of results can be challenging, and resource managers must have a clear understanding of both the strengths and limitations when evaluating eDNA-based monitoring data.  To assess the benefit of a standardized eDNA monitoring approach we work with local stakeholders to collect water samples in coordination with existing long term water quality and fish monitoring programs in 10 estuaries in the United States.   These sites represent very different conditions (e.g., latitude, geomorphology, temperature, salinity, etc.) and aquatic communities.  By applying the same sampling and analysis method at each location we can compare the results from a standardized method to better understand the practical use of eDNA monitoring in estuarine systems. We seek to answer three questions posed by resource managers:  What aquatic species can we detect? How does eDNA-based monitoring compare to traditional fish surveys? And what is the relative cost of these methods?    The project team is comprised of researchers and end users representing multiple state and nonprofit agencies, each with varying levels of experience in molecular methods.   Communication and training within the team is tailored to address individual interests and capacity, with the goal of supporting a national network of end users who are incorporating eDNA into coastal management.

  • Title: The impact of intragenomic rRNA variation on alpha-diversity estimates in metabarcoding studies: a case study from marine nematodes

Affiliation: Department of Marine Sciences, University of Georgia

Email: tiago.pereira@uga.edu

Author: Tiago Jose Pereira

Abstract: Intragenomic rRNA variation is a critical concern for eukaryotic metabarcoding studies, due to its potentially confounding effects on species delimitation and biodiversity estimates derived from -Omics data. In the present study, we assessed patterns associated with 18S rRNA metabarcoding loci in marine nematodes, including characterization of intragenomic rRNA gene variants (number of variants and abundance profiles) and aspects of datasets that can obscure biological signals (e.g., amplification of non-target DNA, ambiguous taxonomy assignments). We estimated Amplicon Sequence Variants (ASVs) using DADA2 from an 18S rRNA metabarcoding dataset (Illumina MiSeq) generated from individual marine nematodes. Illumina data were analyzed in conjunction with nematode morphological identifications and nearly full-length 18S reference sequences (~1600bp Sanger barcodes) generated for a subset of the same specimens. Our results indicated that levels of intragenomic rRNA variation appeared to vary widely across nematode taxa (irrespective of phylogenetic clades or ecological feeding groups), and that co-amplification of non-target DNA was common (relic DNA, gut contents, etc.). The DADA2 pipeline appeared to produce a biologically accurate profile of intragenomic rRNA variants in nematodes that was consistent with “Head-Tail” patterns (of dominant vs. minor rRNA gene variants) identified in previous studies. Although intragenomic rRNA variation appears to be ubiquitous in marine nematodes, nematode identifications were highly congruent across our three methods for species delimitation (traditional morphological taxonomy, Sanger DNA barcoding, and high-throughput metabarcoding). In spite of pervasive intragenomic variation and high copy number of rRNA genes, the most abundant ASVs in metabarcoding datasets are likely to represent true species barcodes and thus confer an accurate view of extant biodiversity. However, our findings also emphasize the importance of applying bioinformatic filtering techniques and developing well-curated reference databases in order to better link rRNA molecules with specimen-level data and alleviate the confounding effects of intragenomic gene variants in studies of microbial eukaryotes.

  • Title: Efficient codetection of diverse targeted species using multiplex environmental DNA assays

Affiliation: Cornell University

Email: jaa53@cornell.edu

Author: Jose Andrés

Abstract: Current environmental DNA (eDNA) assays offer the possibility of either single-species detection (e.g., qPCR, ddPCR, CRISPR-Cas) or the characterization of whole communities using metabarcoding. But, single-species methods may become prohibitively expensive when there is a need to target multiple species, and metabarcoding bioinformatics analyses are prohibitively complex for many management scenarios. To address these challenges, we developed a cost-effective, standardizable, multiple-amplicon eDNA method to effectively conduct early detection of multiple targeted species single assays. As proof-of-principle, we successfully tested and applied this method in four of NY’s Great Lakes harbors where new invasive species are likely to become established and in New York City’s harbor, where multiple marine species are known to exist. The first assay was optimized to codetect 17 Great Lakes invasive species, while the second one targeted 10 New York harbor species. Species successfully simultaneously targeted include both plants and animals. Several invasive species of interest to managers in the NY Great Lakes basin and throughout North America were detected (F. rusticus, N. obtusa, D. polymorpha, and N. melanostomus). The general protocol is highly specific and is applicable in ecosystems worldwide.

  • Title: Genetic variation in environmental samples as a metric of species’ abundance.

Affiliation: Cornell University

Email: jaa53@cornell.edu

Author: Jose Andrés

Abstract: Most efforts to assess species abundance with environmental DNA (eDNA) have focused on correlating eDNA concentration (assessed by qPCR) with estimates of numerical abundance. However, such relationship may be confounded by variation in eDNA concentration due sampling stochasticity, and complex factors influencing DNA production and loss rates.  A possible solution to overcome these issues is to apply analytical frameworks that incorporate information about the genetic diversity present in eDNA samples. While we have successfully used DNA mixture models to estimate fish abundance in mesocosms where the total number of individuals is small (≤ 10), the limitations of this method has not been thoroughly explored. To advance our understanding of the performance of DNA mixture models in ecological and environmental DNA applications, we explored abundance estimation in simulated mixtures of DNA made up of larger numbers of putative contributors that may reflect local abundances experienced in ecological studies. We find that DNA mixture models can estimate the number of individuals contributing to mixtures of DNA of up to 100 individuals, provided enough genetic diversity and rare alleles are present in the sample. The presence of rare alleles in a DNA mixture is more important than the number of loci for accurately estimating the number of contributing individuals, and microsatellites or mitochondrial haplotypes outperform bi-allelic SNPs because the per-locus information content is much higher.

  • Title: Seafood Tracer’s mobile eDNA lab kit and eDNA collection device for seafood traceability

Affiliation: Loyola Marymount University

Email: demian.willette@lmu.edu

Author: Demian Willette

Abstract: Traceability is a persistent challenge throughout the seafood supply chain, and increasingly complex and consequential as seafood consumption and illegal fishing operations have increased globally. Catalyzed through the National Science Foundation Convergence Accelerator program, we developed Seafood Tracer, a tool that leverages environmental DNA (eDNA), computer vision AI, and species spatial distribution to deliver near real-time species identification to seafood supply chain end-users through a web-based application. Of particular interest to this eDNA workshop audience, we designed and field-tested two patent-pending 3D-printed passive eDNA collection devices – one for stationary sampling in low-flow settings and another for dynamic sampling on moving vessels or in other high-flow conditions. Use-inspired design was applied to create intuitive and functional features. We tested different filter membranes of varied composition and pore size to determine the most cost-effective and suitable membrane for downstream DNA extraction methods using commercially available spin column kits. To process samples, we assembled and field-tested our mobile eDNA lab kit, a fully battery-powered, portable lab that fits into two storm cases. Processing time from eDNA sample collection to sequence data generation amounts to just under 10 hours. Furthermore, eDNA collection devices are printed using conditionally compostable materials, which include both a plant-based Polyactic Acid (PLA) filament and an algal-based PLA filament to establish optimal durability over multiple deployments. Many of the initial objectives of this project have been reached, including detecting universal gene targets as eDNA in controlled lab and open ocean settings, signaling the readiness of the technology to be employed at scale, in support of transparency in the seafood supply chain, a key requirement for sustainable Blue Economy.

  1. Title: Integrating and harmonizing molecular observations in support of sustainable marine management through the proposed Southern California Ocean Biomolecular Observing Network (SoCal-OBON)

Affiliation: CalCOFI & California Sea Grant, Scripps Institution of Oceangraphy, UCSD

Email: esatterthwaite@ucsd.edu

Authors: Erin Satterthwaite

Coauthors: Zachary Gold*, CalCOFI/SCCWRP; Brice Semmens, CalCOFI, Scripps Institution of Oceanography, UCSD; Noelle Bowlin, CalCOFI, NOAA Southwest Fisheries Science Center; Susie Theroux, SCCWRP; Clarissa Anderson, SCCOOS, Scripps Institution of Oceanography, UCSD; Mark Ohman, CCE-LTER, Scripps Institution of Oceanography, UCSD; Stephanie Matthews- CCE-LTER, Scripps Institution of Oceanography, UCSD

Abstract: Biomolecular technologies offer significant potential to improve and transform marine management, but the development and application of molecular tools in marine ecosystems has been siloed within disciplines (e.g., microbial ecology, phycology, fisheries), over space (e.g., nearshore versus offshore), and across sectors (regulatory, academic, industry). This has led to isolated advances and slow adoption of molecular tools for marine biomonitoring in support of sustainable marine management. To break down these barriers and advance the implementation of molecular tools for marine biomonitoring, we are proposing the creation of the Southern California Ocean Biomolecular Network (SoCal-OBON) within the context of the international process, the UN Decade of Ocean Science for Sustainable Development (2021- 2030).

The SoCal-OBON will be a cooperative of multiple southern California ocean observing programs and regional ocean stakeholders collectively promoting the integration and harmonization of molecular approaches in the Southern California regional biomonitoring efforts. The Cooperative’s main objective is to provide an unprecedented resolution of key ecological indicators across space, time, and biodiversity relevant to application, use, and management. The proposed Cooperative will: 1) identify gaps to interoperability of ocean observing platforms; 2) conduct molecular intercalibration efforts, and 3) establish best practices for large scale biomolecular monitoring. We hope that SoCal-OBON will serve to provide guidance on best practices and effectively integrate molecular observations off the coast of California and beyond. 

  1. Title: Assessing seasonal occupancy of winter flounder (Pseudopleuronectes americanus) in Massachusetts embayments using eDNA

Affiliation: Gloucester Marine Genomics Institute

Email: tim.odonnell@gmgi.org

Author: Tim O’Donnell

Abstract: Dredging projects are vital to maintain and improve navigational waterways that support the economic growth and sustainability of coastal communities. However, dredging can disturb wildlife habitats and result in detrimental consequences for economically important fish species like winter flounder (Pseudopleuronectes americanus) that use coastal embayments as spawning habitat. Turbidity and burial caused by dredging can lead to mortality of demersal eggs, which has prompted conservative dredging restrictions to protect winter flounder spawning success for up to six months of the year due to a lack of detailed information regarding the timing and location of winter flounder spawning within embayments. To better understand winter flounder occupancy patterns in coastal Massachusetts, we have initiated an eDNA study by filtering (0.2µm pore size) 10-13 1L water samples across six embayments each month over a year, extracting DNA with the Qiagen DNeasy PowerSoil Pro kit, and screening for the presence of winter flounder with a species-specific qPCR assay. Preliminary results indicate seasonal and embayment-specific trends in winter flounder occupancy likely driven by benthic habitat and differences in behaviors among adult and young-of-year life stages. Completed data will be used to build an occupancy model to inform dredging regulations that protect the winter flounder fishery while potentially identify times and/or regions where restrictions can be relaxed.

  1. Title: Assessing marine mammal distribution in the New York Bight using eDNA metabarcoding

Affiliation: California State University, Monterey Bay

Email: ealter@csumb.edu

Author: Liz Alter

Abstract: Determining how cetaceans and other threatened marine animals use coastal habitats is critical to the effective conservation of these species over broad spatial and temporal scales. eDNA may present a useful complementary method for monitoring marine mammal presence during visual surveys in nearshore areas, and for co-detecting prey. In conjunction with ongoing visual surveys, we tested the ability of eDNA metabarcoding to detect the presence and identity of cetaceans in the New York Bight (NYB), and to identify fish species (potential prey) present in the area. In almost all cases in which humpback whales and dolphins were visually observed, DNA from these species was also detected in water samples. To assess eDNA degradation over time, we took samples in the same location 15 and 30 minutes after a sighting in seven instances, and found that eDNA often, but not always, dropped to low levels after 30 minutes. Atlantic menhaden were detected in all samples and comprised the majority of fish sequences in most samples, in agreement with observations of large aggregations of this important prey species in the NYB. While additional data are needed to better understand how factors such as behavior and oceanographic conditions contribute to the longevity of eDNA signals, these results add to a growing body of work indicating that eDNA is a promising tool to complement visual and acoustic surveys of marine megafauna.

  1. Title: Community consensus development of environmental DNA methodological standards for the Nonindigenous Aquatic Species Database

Affiliation: U.S. Geological Survey

Email: mhunter@usgs.gov

Author: Maggie Hunter

Abstract: Environmental DNA (eDNA) detection in water samples is a powerful tool to detect the spread and distribution of aquatic invasive species. The U.S. Geological Survey Nonindigenous Aquatic Species database (NAS) displays visual observation and collection data to improve Early Detection and Rapid Response efforts (nas.er.usgs.gov). Here, we led an effort to include eDNA data and metadata alongside traditional sighting data in marine, freshwater and brackish ecosystems. To publicly display this sensitive and analytically powerful eDNA data, proper sampling, analytical procedures and careful validation are required for accurate interpretation and to avoid false detections and/or non-detections. The eDNA community was engaged to develop best practices and standards by working with practitioners and natural resource managers to reach consensus on a submission application and data template. This documentation was developed to verify that critical experimental controls and standard methods that were employed to produce the data. Virtual town halls with resource managers, regular meetings by a core scientific advisory panel, and outreach to the broader eDNA community allowed for iterative reviews and integral feedback to establish a community consensus on these developed minimum standards. Additionally, a communication plan was established to inform partners and managers of submitted or displayed eDNA detection data within their jurisdictions. The comprehensive best practices and data standards developed here for database display of eDNA data are now under review for broader application in the field.

  1. Title: (Re)calibrating eDNA metabarcoding for measuring fish species richness in marine ecosystems

Affiliation: University of California, Santa Barbara

Email: mcelroy@ucsb.edu

Author: Mary McElroy

Abstract: This poster presents an updated calibration of eDNA metabarcoding to conventional fish diversity surveys (visual census, capture, etc.) and a global synthesis of eDNA metabarcoding’s relative performance for measuring fish species richness and community composition across diverse marine ecosystems. After identifying 35 publications based on literature review, we performed agreement analysis (Lin’s concordance correlation) and Bland-Altman visualization to assess the extent to which eDNA metabarcoding and conventional surveys detect the same numbers and kinds of fish species at shared sampling locations (n=41) ranging from deep polar seas to tropical coral reefs. We also evaluated relative performance for single- vs. multi-marker eDNA metabarcoding and explored whether fish species undetected by eDNA lacked genetic reference sequences. Overall, we found eDNA metabarcoding performs as well as conventional surveys in marine systems for measuring fish species richness, and multi-marker assays improve the approach’s performance. At all sites, there was some degree of shared species detection but also a high degree of complementary detection with both methods identifying unique species. For a subset of single-marker studies (n=17), eDNA estimates of fish species richness could potentially improve by 20% on average with genetic reference development for 187 unsequenced species. Expanding eDNA metabarcoding applications in marine systems show how its performance for fish diversity assessment has improved significantly over a very short period of time.

  1. Title: Using Environmental DNA to Characterize Marine Community Composition Across Spatial and Environmental Gradients in Southern California Kelp Forests

Affiliation: UCLA

Email: mauraeva@gmail.com

Author: Maura Palacios Mejia

Abstract: The coasts of Southern California are home to giant kelp forests (Macrocystis pyrifera), highly productive ecosystems that foster high levels of biodiversity. Kelp forests vary in chemical and physical properties across both horizontal and vertical gradients, driving the community composition of marine organisms. Environmental DNA (eDNA) metabarcoding is a new method to assess marine community composition and  in combination with environmental variables can provide insights on marine biogeographic patterns. This study explores the community composition along spatial and environmental gradients in three Southern California Kelp Forests. A total of 24 seawater eDNA samples were collected at 3 different depths across 3 kelp forests in summer 2021. We amplified for fish (12S primer), marine algae and invertebrates (COI primer) and modeled community composition by region, depth, and environmental variables from Bio-ORACLE. We find significant differences in community composition across regions for fishes, algae, and invertebrates, but not depth, and we identified specific species associated with each kelp forest site. However, there was more spatial variation in algal and invertebrate communities than in fish communities. We found a significant effect of chlorophyll-a in driving community variation, but this effect was ultimately masked by spatial variation. We also found a significant interaction between spatial variation and dissolved oxygen in driving community variation. Ultimately, our study indicates that eDNA metabarcoding is effective in characterizing marine biogeographic patterns on a local and regional scale.

  1. Title: WANTED: Searching for Longfin Smelt in the Nooksack Estuary and Bellingham Bay Affiliation: Northwest Indian College

Email: rmallon@nwic.edu

Author: Rachael Mallon

Abstract: Longfin Smelt (Spirinchus thaleichthys; LFS) is a small forage fish that are present from San Francisco Bay, California, USA to Prince William Sound, Alaska. The well-studied population from San Francisco Bay area are considered threatened under the California Endangered Species Act due to population decline, which is a concern to the people who rely on this fish for subsistence and cultural needs. Lummi people and Pacific Northwest elders similarly describe a decline of LFS on the Nooksack River near Bellingham, WA where there is an annual spawning run of this anadromous fish. However, the life cycle of LFS is poorly characterized. This study uses a novel high-sensitivity species-specific TaqMan quantitative PCR (qPCR) assay to detect environmental DNA (eDNA) of LFS in marine and freshwater. eDNA is released by organisms in their environment, which can be used for the detection of species by collecting less-invasive water samples. This study was used to investigate the spawning habitat and lifecycle of LFS in Bellingham Bay, WA, USA. Using this assay we analyzed four years of data and to detect LFS at six successive sites 0-6 miles upriver from Bellingham Bay in the Nooksack River. Additionally, a preliminary survey of Bellingham Bay and the Nooksack estuary detects LFS for many months following spawning. This fishery is co-managed by tribal (Lummi Natural Resources) and State (Washington Department of Fish and Wildlife) entities and additional information regarding LFS spawning, rearing, and migration dynamics could inform management practices of this culturally important population.

  • Title: Thinking of eDNA? Guidance on eDNA for management in marine ecosystems

Affiliation: Natural History Museum of Los Angeles County

Email: pentcheff@nhm.org

Author: Regina Wetzer

Coauthors: N. Dean Pentcheff, Adam Wall, Zachary Gold

Abstract: Environmental DNA (eDNA) metabarcoding uses genetic sequences from environmental samples to inventory biological diversity. Genetic material from organismal DNA shed into the environment can be extracted, sequenced, and matched against reference sequences to yield a list of taxa that were in the area where the sample was taken. Sampling and sequencing a cup of seawater is dramatically cheaper than conventional biological surveying, particularly in the ocean, prompting increasing interest in eDNA metabarcoding for environmental monitoring and management applications. But, like every method, eDNA metabarcoding requires careful study design. We explored aspects of eDNA study design in a case study using eDNA in a highly urban marine setting (the Port of Los Angeles and Port of Long Beach), helping to develop a set of recommendations for future studies.

  • Title: Aquatic Biosurveillance using eDNA/eRNA: advantages and challenges 

Affiliation: University of Michigan

Email: chaganti@umich.edu

Author: Subba Rao Chaganti

Abstract: Using environmental DNA (eDNA) for monitoring aquatic species has rapidly gaining interest across the globe. However, eDNA methodology still needs to be stream lined to avoid the concerns from the legacy DNA that could misrepresent the aquatic species distribution. These drawbacks often raises major concerns for the managers or for the stakeholders for decision making. We showcase how environmental RNA (eRNA) could be paired up with the eDNA to avoid the legacy eDNA issues and how to avoid the major challenges involved in eRNA sample collection and processing.

Affiliation: Johns Hopkins University Applied Physics Laboratory

Email: kristina.zudock@jhuapl.edu

Author: Kristina Zudock

Coauthors: H.M. DeHart, N. Brown, W.J. Ross1, C. Li, and P. Thielen

Abstract: There are twenty-one families which comprise marine mammals across the world’s seas and oceans. Across these families, there exist markedly different population abundances, rates of intra-species diversity, and availability of public genomic references. In order to rapidly determine the presence of marine mammals in the environment, we have developed a fieldable eDNA analysis pipeline which utilizes the K-mer-based classifier Kraken2 along with bespoke reference databases for mitochondrial and ribosomal amplicons to analyze Oxford Nanopore runs within seconds using only a laptop and/or low-energy GPU. While single references per species is often standard in compact reference databases for amplicon taxonomic classification, we have found that in the case of recently-diverged lineages, such as those in Delphinidae, multiple references per species are essential to cover the intra-species diversity and reduce misclassifications. However, a reference database which contains an overabundance of sequences for certain taxa over others also has the possibility of increasing false positives for the overrepresented taxa. Thus, we have explored the essential balance of over- versus under-representation of species in reference databases for K-mer classification of amplicons and have identified lineages where the availability of public reference data would benefit to be expanded in order to increase the sensitivity and specificity of eDNA-based marine mammal identification.

Affiliation: Johns Hopkins APL

Email: Tobias.dilworth@jhuapl.edu.

Author: Tobias Dilworth

Abstract: Traditionally, aquatic environmental DNA (eDNA) characterization requires sample collection at a field site, capture of suspended biological matter on a filtration membrane, purification of DNA from said filter, and analysis of the sample using one or more molecular approaches. This process is logistically complex, and often requires many days to months between sample collection and analysis. Our group is currently developing an end-to-end pipeline for the complete automation of eDNA sample collection and analysis, with the end goal of producing data in situ aboard an unmanned underwater vehicle (UUV). This presentation will discuss progress towards sample processing automation using a bespoke microfluidics platform designed for targeted amplicon sequencing (e.g. metabarcoding) and capable of producing a sequence-ready product for the Oxford Nanopore Technologies MinION sequencer.