Emerging Methods for Detection and their Potential for Inventorying Brook Trout Populations in Streams of the Western Adirondacks
Background
Deoxyribonucleic acid (DNA) is the hereditary material in organisms that contains the biological instructions for building and maintaining them. The chemical structure of DNA is the same for all organisms, but differences exist in the order of the DNA building blocks, known as base pairs. Unique sequences provide a means to identify individual species and detect their presence within aquatic or terrestrial environments. Environmental DNA (eDNA) is nuclear or mitochondrial DNA that is shed from an organism into the environment. Sources of eDNA include feces, mucous, and gametes; shed skin; and carcasses. In aquatic environments, eDNA is diluted and distributed by currents and other hydrological processes. It may remain in the environment from days to millennia, depending on conditions and sampling procedures and conditions (Dejean et al. 2011; Epp et al. 2010).
Environmental DNA analysis is a newly emerging and rapidly evolving molecular-genetics tool with a large capacity to help effectively monitor, manage, and conserve fishery resources. Environmental DNA can be detected using routine molecular techniques such as the polymerase chain reaction (PCR) to amplify species-specific genes, thus, detecting their presence without actually observing them. Species detections, however, can be difficult in many environments and in populations at low densities and DNA detectability decreases with time after the removal of the species source of DNA (Dejean et al. 2011). Thus, the dynamics of detectability reflect the persistence of DNA fragments in freshwater systems. Environmental DNA analysis is currently being evaluated for uses such as surveillance and control of aquatic invasive species, identification and monitoring of endangered species, and analysis of biodiversity. For species that are rare or difficult to find, the methods can enhance detection, which improves biodiversity assessments. Because all aquatic organisms naturally shed DNA into water they occupy, eDNA can be analyzed to monitor the presence or absence and relative abundance of individual species and richness of species assemblages in aquatic ecosystems.
The historic distribution of eastern brook trout (Salvelinus fontinalis) and diversity of native fish species have decreased steadily in streams and lakes across the northeast over the past 100 years due to many stressors; however, the losses of brook trout and other fish-species populations have been precipitous in poorly buffered and remote regions of the Adirondacks due to acidic deposition. Over the past 10-15 years, water quality may have improved to the point that acid-tolerant brook trout and other native species could be reestablished in formerly acidified streams and lakes due to implementation of the Clean Air Act of 1990 and reduced emissions of sulfur (SOx) and nitrogen (NOx) oxides, decreased acidic deposition, and decreased surface-water toxicity. Increases in DOC levels in humic systems may also be impeding formation of toxic inorganic Al (Alim) fractions, which further improves water quality. No systematic state or federal program currently exists to monitor and assess recovery of fish populations in surface waters of the Adirondack, partly because few tools are available to inexpensively and accurately detect the presence or absence of brook trout, or other game and non-game species, in remote or easily accessible streams and rivers of the region.
Objective
The primary objectives of this study are to (a) assess and refine eDNA methods for detecting the presence or absence (and relative abundance) of eastern brook trout and (b) determine the present-day status, relative abundance, and distribution of brook trout in headwater streams of the western Adirondacks. A related goal of this study is to assess the effectiveness (cost and time) of eDNA as a standardized tool for rapid and cost-effective collection of data that describes the status and distribution of brook trout in the Adirondacks. Although brook trout are the primary target for this study, the capacity and cost of eDNA to detect the presence or absence, and relative abundance of other native and invasive fish species will be informally assessed.
Results of this study address several Strategic Objectives listed in the NYSERDA’s recent Environmental Research Plan for Research Area 1: Ecological Effects of Deposition of Sulfur, Nitrogen, and Mercury (NYSERDA 2013). The study will evaluate the cost, accuracy, and effectiveness of an new/emerging molecular tool (eDNA) to survey and monitor the presence/absence, relative abundance, and distribution of one or more fish species in surface waters (streams and lakes) of the Adirondacks. Thus, the study directly addresses NYSERDA’s Strategic Objective 10 (Ecosystem Management). The proposed research and analyses will also help define the status of brook trout populations in streams of the western Adirondacks that are recovering from decades of acidification. Thus, by defining the response of brook trout (increases in population density and in their distribution) to improved water quality and the implications for recovery of entire fish assemblages and stream ecosystems due to SOx and NOx emission reductions in the Adirondacks, this study addresses NYSERDA’s Strategic Objective 2 (Ecological Effects of Deposition of Sulfur, Nitrogen, and Mercury). The information generated describing the present-day distributions of brook trout, combined with water chemistry (e.g., Al fractions, pH, ANC, BCS, Ca, and DOC) data from ongoing Western and East-central Adirondack Stream Survey (WASS and ECASS) projects will provide measures of potential recovery (ecosystem response) in some of the most acid sensitive streams in the Adirondack region and help link current SOx and NOx emission levels to biological responses, which can inform the USEPA’s process that is developing secondary National Ambient Air Quality Standards (NAAQS) for oxides of nitrogen (NOx) and oxides of sulfur (SOx) and NYSERDA’s Strategic Objective 3 (Multi-Media Monitoring and Trend Analysis for Sulfur and Nitrogen).
Approach
The U.S. Geological Survey (USGS) in cooperation with the Paul Smith’s Adirondack Watershed Institute (AWI) propose a 2-year study to assess and refine eDNA methods for detecting the presence or absence (and relative abundance) of eastern brook trout and assess the current status, relative abundance, and distribution of brook trout in headwater streams of the western Adirondacks. The potential of eDNA as a standardized tool for rapid and cost-effective monitoring and assessment of resident fish species will also be evaluated.
Task 1, Fish surveys and fish-tissue samples: The composition of fish communities, the presence of brook trout populations, and water chemistry in as many as 30 stream reaches in the western Adirondacks, will be defined (at no cost to the proposed study) through quantitative surveys of fish assemblages during 2014 and 2015 as part of an independent stream-fish study (Monitoring effects of acidification and mercury on fish in New York streams, with special emphasis on the Adirondack Ecoregion), managed by B. Baldigo and K. Riva-Murray (USGS-NYWSC, Troy, NY). During these surveys, fin clip samples from brook trout (and other common species) will be collected, preserved in ethanol, and forwarded to the AWI. All collected tissues will be archived and those from brook trout will subsequently be processed to purify and quantitate total genomic DNA using the DNeasy genomic DNA purification kit (Qiagen™). Quantitation and assessment of purity will be performed using a NanoDrop spectrophotometer housed at the Trudeau Institute, Saranac Lake, NY. Total genomic DNA obtained from these tissues will be used to create standard curves for development of efficient and species-specific quantitative (real time) PCR tests.
Task 2, Develop species-specific qPCR test: Quantitative, real-time (Taqman™) polymerase chain reaction (PCR) will be used to develop a test that detects a portion of the DNA barcode gene (mitochondrial cytochrome oxidase subunit 1) that is specific for brook trout in environmental samples (Hajibabaei et al. 2006; Hebert et al. 2003). Sequences contained in GenBank™ have been downloaded and primer and probe combinations designed using Beacon Designer™ software (Premier Biosoft), which optimizes for intended targets, reduces chances of cross-reactivity with unintended target species and eliminates regions of template secondary structure. The protocol for detection will be optimized using total genomic DNA from the species of interest, prior to testing of environmental samples. In addition, species-specificity will be assessed using total genomic DNA obtained from a variety of non-target fish species. Sensitivity of detection will be assessed using serial dilution of template, and possible interference by environmental contaminants will be controlled for by spiking environmental water and sediment samples with known amounts of template DNA, followed by recovery and PCR detection.
Task 3, Assess sampling methods: The experience of some researchers has shown that the sampling process can affect their ability to accurately detect the presence/absence (or relative abundance) of single or multiple species (Mahon et al. 2013; Pilliod et al. 2013). Thus, water will be collected from as many as 12 stream reaches (fall 2014, winter 2014-15, and spring 2015) where brook trout populations were found to be absent or exist in low, moderate, and high densities using several collection methods. Additional sediment samples will be collected from half of the sites to determine if sediment might be a better medium to obtain eDNA for stream fish. All water samples will be assessed using the revised/refined brook trout primers to assess method effectiveness and to determine which provides the most accurate determination of their presence (or absence) and relative abundances of their populations (up to 12 sites, sampled 3 times = 36 water samples, 12 sediment samples).
Task 4, Water collections: Two-liter water samples will be collected once from each of 30 study sites in the Adirondacks where brook trout densities were quantified during 2014-15. All samples will be preserved on ice, filtered onto 1.5 micron pore size glass microfiber filters within 6 hours of collection, then frozen and transported to AWI for purification using the PowerWater™ DNA purification kit (Mo Bio Laboratories) and eDNA analysis.
Task 5, eDNA sample analysis: Environmental DNA will be purified from all water or sediment samples by the AWI using the PowerWater™ or PowerSoil™ DNA purification kits following manufacturer’s instructions, quantitated and submitted for real-time PCR analysis. The procedure involves emulsification of the glass fiber filter by agitation with garnet beads, followed by precipitation of contaminants and concentration of eDNA using a silica gel membrane. Following quantitation of DNA, samples will be submitted for species-specific detection by qPCR.
Task 6, Assess data and relations: The accuracy of eDNA results to predict the presence/absence and the relative abundance of brook trout will be assessed using two methods and all eDNA data from the 30 streams where fish assemblages were sampled during 2014-15. A logistic regression model will be used to determine how well an eDNA-probability model predicts the presence or absence of brook trout at all 30 study reaches. A linear or curvilinear regression model will be used to determine the ability and accuracy of eDNA “counts” or relative abundance to predict the known (measured) density of brook trout populations.
Task 7, Prepare final report/paper: The USGS and AWI will compile and summarize project results in a final report or journal article that (1) defines the eDNA methodology and efficacy for detecting the presence or absence, and relative abundance, of eastern brook trout, (2) assesses how accurately eDNA characterizes the present-day status, relative abundance, and distribution of brook trout in headwater streams (compared to routine electrofishing surveys), and (3) evaluates the actual and potential effectiveness (cost and effort) of eDNA as tool to monitor and assess the health and distribution of brook trout (and other fish species) populations in streams of the western Adirondacks. The paper or report will go through internal technical and editorial reviews, be revised, and then be submitted to a peer-reviewed journal or published as a NYSERDA report. In addition, we will prepare a brief summary paper, approximately four to eight pages in length, which translates scientific findings into a fashion that is interesting, understandable, and appealing to a broad audience, including policy analysts, policy makers, and the interested general public.
Report
Baldigo, Barry P., Sporn, Lee Ann, George Scott D., and Ball, Jacob. 2016. Efficacy of environmental DNA to detect and quantify Brook Trout populations in headwater streams of the Adirondack Mountains, New York, Transactions of the American Fisheries Society, Volume146, Issue1, January 2017, Pages 99-111
Project
Location by County
Lewis County, NY, St. Lawrence County, NY, Hamilton County, NY, Herkimer County, NY, Oneida County, NY
- Source: USGS Sciencebase (id: 55df247be4b0518e354e093c)
Background
Deoxyribonucleic acid (DNA) is the hereditary material in organisms that contains the biological instructions for building and maintaining them. The chemical structure of DNA is the same for all organisms, but differences exist in the order of the DNA building blocks, known as base pairs. Unique sequences provide a means to identify individual species and detect their presence within aquatic or terrestrial environments. Environmental DNA (eDNA) is nuclear or mitochondrial DNA that is shed from an organism into the environment. Sources of eDNA include feces, mucous, and gametes; shed skin; and carcasses. In aquatic environments, eDNA is diluted and distributed by currents and other hydrological processes. It may remain in the environment from days to millennia, depending on conditions and sampling procedures and conditions (Dejean et al. 2011; Epp et al. 2010).
Environmental DNA analysis is a newly emerging and rapidly evolving molecular-genetics tool with a large capacity to help effectively monitor, manage, and conserve fishery resources. Environmental DNA can be detected using routine molecular techniques such as the polymerase chain reaction (PCR) to amplify species-specific genes, thus, detecting their presence without actually observing them. Species detections, however, can be difficult in many environments and in populations at low densities and DNA detectability decreases with time after the removal of the species source of DNA (Dejean et al. 2011). Thus, the dynamics of detectability reflect the persistence of DNA fragments in freshwater systems. Environmental DNA analysis is currently being evaluated for uses such as surveillance and control of aquatic invasive species, identification and monitoring of endangered species, and analysis of biodiversity. For species that are rare or difficult to find, the methods can enhance detection, which improves biodiversity assessments. Because all aquatic organisms naturally shed DNA into water they occupy, eDNA can be analyzed to monitor the presence or absence and relative abundance of individual species and richness of species assemblages in aquatic ecosystems.
The historic distribution of eastern brook trout (Salvelinus fontinalis) and diversity of native fish species have decreased steadily in streams and lakes across the northeast over the past 100 years due to many stressors; however, the losses of brook trout and other fish-species populations have been precipitous in poorly buffered and remote regions of the Adirondacks due to acidic deposition. Over the past 10-15 years, water quality may have improved to the point that acid-tolerant brook trout and other native species could be reestablished in formerly acidified streams and lakes due to implementation of the Clean Air Act of 1990 and reduced emissions of sulfur (SOx) and nitrogen (NOx) oxides, decreased acidic deposition, and decreased surface-water toxicity. Increases in DOC levels in humic systems may also be impeding formation of toxic inorganic Al (Alim) fractions, which further improves water quality. No systematic state or federal program currently exists to monitor and assess recovery of fish populations in surface waters of the Adirondack, partly because few tools are available to inexpensively and accurately detect the presence or absence of brook trout, or other game and non-game species, in remote or easily accessible streams and rivers of the region.
Objective
The primary objectives of this study are to (a) assess and refine eDNA methods for detecting the presence or absence (and relative abundance) of eastern brook trout and (b) determine the present-day status, relative abundance, and distribution of brook trout in headwater streams of the western Adirondacks. A related goal of this study is to assess the effectiveness (cost and time) of eDNA as a standardized tool for rapid and cost-effective collection of data that describes the status and distribution of brook trout in the Adirondacks. Although brook trout are the primary target for this study, the capacity and cost of eDNA to detect the presence or absence, and relative abundance of other native and invasive fish species will be informally assessed.
Results of this study address several Strategic Objectives listed in the NYSERDA’s recent Environmental Research Plan for Research Area 1: Ecological Effects of Deposition of Sulfur, Nitrogen, and Mercury (NYSERDA 2013). The study will evaluate the cost, accuracy, and effectiveness of an new/emerging molecular tool (eDNA) to survey and monitor the presence/absence, relative abundance, and distribution of one or more fish species in surface waters (streams and lakes) of the Adirondacks. Thus, the study directly addresses NYSERDA’s Strategic Objective 10 (Ecosystem Management). The proposed research and analyses will also help define the status of brook trout populations in streams of the western Adirondacks that are recovering from decades of acidification. Thus, by defining the response of brook trout (increases in population density and in their distribution) to improved water quality and the implications for recovery of entire fish assemblages and stream ecosystems due to SOx and NOx emission reductions in the Adirondacks, this study addresses NYSERDA’s Strategic Objective 2 (Ecological Effects of Deposition of Sulfur, Nitrogen, and Mercury). The information generated describing the present-day distributions of brook trout, combined with water chemistry (e.g., Al fractions, pH, ANC, BCS, Ca, and DOC) data from ongoing Western and East-central Adirondack Stream Survey (WASS and ECASS) projects will provide measures of potential recovery (ecosystem response) in some of the most acid sensitive streams in the Adirondack region and help link current SOx and NOx emission levels to biological responses, which can inform the USEPA’s process that is developing secondary National Ambient Air Quality Standards (NAAQS) for oxides of nitrogen (NOx) and oxides of sulfur (SOx) and NYSERDA’s Strategic Objective 3 (Multi-Media Monitoring and Trend Analysis for Sulfur and Nitrogen).
Approach
The U.S. Geological Survey (USGS) in cooperation with the Paul Smith’s Adirondack Watershed Institute (AWI) propose a 2-year study to assess and refine eDNA methods for detecting the presence or absence (and relative abundance) of eastern brook trout and assess the current status, relative abundance, and distribution of brook trout in headwater streams of the western Adirondacks. The potential of eDNA as a standardized tool for rapid and cost-effective monitoring and assessment of resident fish species will also be evaluated.
Task 1, Fish surveys and fish-tissue samples: The composition of fish communities, the presence of brook trout populations, and water chemistry in as many as 30 stream reaches in the western Adirondacks, will be defined (at no cost to the proposed study) through quantitative surveys of fish assemblages during 2014 and 2015 as part of an independent stream-fish study (Monitoring effects of acidification and mercury on fish in New York streams, with special emphasis on the Adirondack Ecoregion), managed by B. Baldigo and K. Riva-Murray (USGS-NYWSC, Troy, NY). During these surveys, fin clip samples from brook trout (and other common species) will be collected, preserved in ethanol, and forwarded to the AWI. All collected tissues will be archived and those from brook trout will subsequently be processed to purify and quantitate total genomic DNA using the DNeasy genomic DNA purification kit (Qiagen™). Quantitation and assessment of purity will be performed using a NanoDrop spectrophotometer housed at the Trudeau Institute, Saranac Lake, NY. Total genomic DNA obtained from these tissues will be used to create standard curves for development of efficient and species-specific quantitative (real time) PCR tests.
Task 2, Develop species-specific qPCR test: Quantitative, real-time (Taqman™) polymerase chain reaction (PCR) will be used to develop a test that detects a portion of the DNA barcode gene (mitochondrial cytochrome oxidase subunit 1) that is specific for brook trout in environmental samples (Hajibabaei et al. 2006; Hebert et al. 2003). Sequences contained in GenBank™ have been downloaded and primer and probe combinations designed using Beacon Designer™ software (Premier Biosoft), which optimizes for intended targets, reduces chances of cross-reactivity with unintended target species and eliminates regions of template secondary structure. The protocol for detection will be optimized using total genomic DNA from the species of interest, prior to testing of environmental samples. In addition, species-specificity will be assessed using total genomic DNA obtained from a variety of non-target fish species. Sensitivity of detection will be assessed using serial dilution of template, and possible interference by environmental contaminants will be controlled for by spiking environmental water and sediment samples with known amounts of template DNA, followed by recovery and PCR detection.
Task 3, Assess sampling methods: The experience of some researchers has shown that the sampling process can affect their ability to accurately detect the presence/absence (or relative abundance) of single or multiple species (Mahon et al. 2013; Pilliod et al. 2013). Thus, water will be collected from as many as 12 stream reaches (fall 2014, winter 2014-15, and spring 2015) where brook trout populations were found to be absent or exist in low, moderate, and high densities using several collection methods. Additional sediment samples will be collected from half of the sites to determine if sediment might be a better medium to obtain eDNA for stream fish. All water samples will be assessed using the revised/refined brook trout primers to assess method effectiveness and to determine which provides the most accurate determination of their presence (or absence) and relative abundances of their populations (up to 12 sites, sampled 3 times = 36 water samples, 12 sediment samples).
Task 4, Water collections: Two-liter water samples will be collected once from each of 30 study sites in the Adirondacks where brook trout densities were quantified during 2014-15. All samples will be preserved on ice, filtered onto 1.5 micron pore size glass microfiber filters within 6 hours of collection, then frozen and transported to AWI for purification using the PowerWater™ DNA purification kit (Mo Bio Laboratories) and eDNA analysis.
Task 5, eDNA sample analysis: Environmental DNA will be purified from all water or sediment samples by the AWI using the PowerWater™ or PowerSoil™ DNA purification kits following manufacturer’s instructions, quantitated and submitted for real-time PCR analysis. The procedure involves emulsification of the glass fiber filter by agitation with garnet beads, followed by precipitation of contaminants and concentration of eDNA using a silica gel membrane. Following quantitation of DNA, samples will be submitted for species-specific detection by qPCR.
Task 6, Assess data and relations: The accuracy of eDNA results to predict the presence/absence and the relative abundance of brook trout will be assessed using two methods and all eDNA data from the 30 streams where fish assemblages were sampled during 2014-15. A logistic regression model will be used to determine how well an eDNA-probability model predicts the presence or absence of brook trout at all 30 study reaches. A linear or curvilinear regression model will be used to determine the ability and accuracy of eDNA “counts” or relative abundance to predict the known (measured) density of brook trout populations.
Task 7, Prepare final report/paper: The USGS and AWI will compile and summarize project results in a final report or journal article that (1) defines the eDNA methodology and efficacy for detecting the presence or absence, and relative abundance, of eastern brook trout, (2) assesses how accurately eDNA characterizes the present-day status, relative abundance, and distribution of brook trout in headwater streams (compared to routine electrofishing surveys), and (3) evaluates the actual and potential effectiveness (cost and effort) of eDNA as tool to monitor and assess the health and distribution of brook trout (and other fish species) populations in streams of the western Adirondacks. The paper or report will go through internal technical and editorial reviews, be revised, and then be submitted to a peer-reviewed journal or published as a NYSERDA report. In addition, we will prepare a brief summary paper, approximately four to eight pages in length, which translates scientific findings into a fashion that is interesting, understandable, and appealing to a broad audience, including policy analysts, policy makers, and the interested general public.
Report
Baldigo, Barry P., Sporn, Lee Ann, George Scott D., and Ball, Jacob. 2016. Efficacy of environmental DNA to detect and quantify Brook Trout populations in headwater streams of the Adirondack Mountains, New York, Transactions of the American Fisheries Society, Volume146, Issue1, January 2017, Pages 99-111
Project
Location by County
Lewis County, NY, St. Lawrence County, NY, Hamilton County, NY, Herkimer County, NY, Oneida County, NY
- Source: USGS Sciencebase (id: 55df247be4b0518e354e093c)