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Sea lamprey (Petromyzon marinus) density estimates using environmental DNA surveillance Active

By Upper Midwest Environmental Sciences Center May 10, 2017

Sea lampreys are a species that invaded the Great Lakes presumably following the improvements made to the Welland Canal in 1920. First reported in Lake Erie in 1921, sea lampreys subsequently spread rapidly to the upper Great Lakes and had an established spawning population in all of the upper Great Lakes by 1947 (Applegate 1950). Following their introduction, sea lamprey devastated the commercial and recreational fisheries of the Great Lakes (Lawrie 1970). Sea lamprey populations are currently managed by various control techniques employed by the Great Lakes Fisheries Commission (GLFC). A significant method of control is the use of chemicals to target the larval phase of the lamprey life cycle (Smith and Tibbles 1980). 

Spawning sea lamprey exhibit potamodromous behavior, leaving the open water of the Great Lakes for tributaries where they swim upstream to spawn. The female lamprey lays between 30,000 to 100,000 eggs into a nest while the male simultaneously fertilizes the eggs. The fertilized eggs hatch into ammocoetes about 18-21 days post fertilization. The ammocoetes spend approximately 4-6 years burrowed in the sediment of the stream where they filter feed on detritus and algae (Applegate 1950). This sedentary lifestyle as a filter feeder makes the ammocoete a prime target for chemical control. However, due to limitations in budget, personnel, and overcoming geographic logistics it is not feasible to treat every sea lamprey-infested stream of the Great Lakes. Therefore, the GLFC must prioritize streams according to estimated lamprey abundance, known treatment costs, and available budget.

Individual stream treatment needs are currently determined using a variety of assessment techniques which include semi-quantitative surveys of potential habitat as well as sea lamprey ammocoete abundance. These surveys are combined with expert judgement of streams gained by managers through years of sea lamprey control. This information is then used by managers to prioritize streams for treatment. The assessment techniques are presumed to have low precision. The idea behind the assessments is not to obtain actual population densities, but rather to prioritize streams for treatment in order to achieve the maximum return on investment of sea lamprey killed per dollar spent. Ranking some streams for treatment can be difficult due to stream conditions (MacKenzie et al. 2005; Darling and Mahon 2011). Providing a quantitative assessment using eDNA, which is minimally labor intensive, could assist management with stream prioritization by supplementing traditional assessment techniques. 

Resource agencies have recently adopted molecular surveillance techniques to detect aquatic invasive species (Darling and Mahon, 2011; Lodge et al., 2012; Jerde et al., 2013). One example of a molecular surveillance technique has been the analysis of water samples for the presence of eDNA using polymerase chain reaction (PCR). Recently researchers used eDNA to look for presence or abundance of multi-cellular organisms (Pilliod 2013; Takahara 2012) in water samples. Some aquatic species such as Asian carp are easily detected using eDNA due to their life history, distribution, and DNA shedding characteristics (Kolar et al. 2007; Jerde et al. 2011; Jerde et al. 2013). Conventional polymerase chain reaction (cPCR) techniques target sections of the mitochondrial DNA of the species in question. Conventional and quantitative PCR has been used to monitor for the presence of silver and bighead carp throughout the Chicago Area Waterway System (CAWS) and the Mississippi, Illinois, Wabash and Ohio rivers, and portions of Lake Erie. Detection assays for eDNA are now developed for numerous terrestrial and aquatic species of native or exotic origin (Ficetola et al. 2008; Goldberg et al. 2011; Dejean et al., 2012; Foote et al. 2012; Takahara et al., 2012; Thomsen et al., 2012a; Thomsen et al., 2012b; Egan et al. 2013; Goldberg et al., 2013; Pilliod et al., 2013; Takahara et al. 2013; Piaggio et al. 2014). Environmental DNA is also useful in detecting various species in lotic aquatic environments (Dejean et al. 2011; Jerde et al. 2011; Minamoto et al., 2012; Thomsen et al. 2012a).

Previous researchers have produced sea lamprey-specific eDNA primers and cPCR assays (Gingera et al., 2016). These assays have been optimized and validated to show they can detect the presence of eDNA from spawning and larval sea lamprey in streams. In addition, they have also found positive correlations with larval densities in a laboratory study. The cPCR assays developed by Gingera et al. are effective at determining presence or absence of the sea lamprey in a stream. However, cPCR analysis of eDNA samples can be challenging due to the presence of non-target DNA and non-specific amplification.  In addition to the challenge of interpreting smeared gels with multiple bands, cPCR analysis requires the additional step of sequencing confirmation.

An alternative method to cPCR for eDNA detection is quantitative PCR (qPCR, or real-time PCR, probe-based PCR, fluorescence-based PCR). Quantitative PCR assays have primers that bind to the target DNA template and also use a probe that is specific to the DNA sequence of the species of interest. There has been research to suggest that qPCR technologies allow correlation of eDNA quantity to species density and distribution (Takahara et al. 2012; Thomsen et al. 2012a). These types of assays have greater sensitivity as well as increased species specificity. The fluorescence obtained during qPCR analysis is able to provide quantitative estimates of DNA in a sample without the need for additional gel analysis and genetic sequencing.
The main objective of this research is to determine if sea lamprey eDNA levels correlate with larval and adult abundance. First, we will conduct density trials with known numbers of sea lamprey under highly controlled laboratory settings. Then, we will collect and analyze water samples from actual streams with different densities of lamprey to demonstrate the utility of this tool under more natural settings. In addition to helping managers rank streams for treatment, this information could also be used to estimate the effectiveness of barriers and traps, and provide quantification of adult spawning runs in streams that are not currently trapped. Validated qPCR markers will be used to test detection probabilities and assess our ability to estimate adult and larval sea lamprey abundance in water collected from tanks and streams that contain sea lamprey.

Objective

  1. The goal of this project is to determine if eDNA abundance is be related to lamprey abundance. We will specifically test two hypotheses:

    Hypothesis 1: If adult sea lamprey shed similar amounts of DNA into the environment, then a system with a greater number of sea lamprey will contain great numbers of sea lamprey eDNA.

    Hypothesis 2: If larval sea lamprey shed similar amounts of DNA into the environment, then a system with a greater number of sea lamprey will contain great numbers of sea lamprey eDNA.
Technical assistance in support of Sea Lamprey Control Program
Sources/Usage: Public Domain. View Media Details
(Public domain.)

 

References

Applegate VC. 1950. Natural history of the sea lamprey (Petromyzon marinus) in Michigan. Special scientific report: Fisheries. U.S. Fish and Wildlife Service. 237 pages.

Darling, J.A., and Mahon, A.R., 2011. From molecules to management: adopting DNA-based methods for monitoring biological invasions in aquatic environments. Environ Res 111: 978-988.

Dejean, T., Valentini, A., Duparc, A., Pellier-Cuit, S., Pompanon, F., Taberlet, P., and Miaud, C. 2011. Persistence of environmental DNA in freshwater ecosystems. PLoS ONE 6(8): e23398.

Dejean, T., Valentini, A., Miquel C., Taberlet, P., Bellemain E., and Miaud, C. 2012. Improved detection of an alien invasive species through environmental DNA barcoding: The example of the American bullfrog Lithobates catesbeianus. Journal of Applied Ecology 49: 953-959.

Egan, S.P., Barnes, M.A., Hwang, C.-T., Mahon, A.R., Feder, J.L., Ruggiero, S.T., Tanner, C.E., and Lodge, D.M. 2013. Rapid invasive species detection by combining environmental DNA with Light Transmission Spectroscopy. Conserv. Lett. 6(6): 402-409.

Erickson, R.A., Rees, C.B., Coulter, A.A., Merkes, C.M., McCalla, S.G., Touzinsky, K.F., Walleser, L., Goforth, R.R., Amberg, J.J., 2016. Detecting the movement and spawning activity of bigheaded carps with environmental DNA. Molecular Ecology Resources. 2016 May 1

Ficetola, G.F., Miaud, C., Pompanon, F., and Taberlet, P. 2008. Species detection using environmental DNA from water samples. Biol. Lett. 4: 423-425.

Foote, A.D., Thomsen, P.F., Sveegaard, S., Wahlberg, M., Kielgast, J., Kyhn, L.A., Salling, A.B., Galatius, A., Orlando, L., and Gilbert, M.T.P. 2012. Investigating the potential use of environmental DNA (eDNA) for genetic monitoring of marine mammals. PLoS ONE 7(8): e41781.

Gelman A, Hill J. Data analysis using regression and multilevelhierarchical models. Cambridge: Cambridge University Press; 2007.

Gingera TD, Steeves TB, Boguski DA, Whyard S, Li Weiming, Docker MF. 2016. Detection and identification of lampreys in Great Lakes streams using environmental DNA. Journal of Great Lakes Research. 42: 649-659.

Goldberg, C.S., Pilliod, D.S., Arkle, R.S., and Waits, L.P. 2011. Molecular detection of vertebrates in stream water: A demonstration using rocky mountain tailed frogs and Idaho giant salamanders. PLoS ONE 6(7): e22746.

Goldberg, C.S., Sepulveda, A., Ray, A., Baumgardt, J., Waits, L.P., 2013. Environmental DNA as a new method for early detection of New Zealand mudsnails (Potamopyrgus antipodarum). Freshwater Science, 32(3): 792-800.

Jerde, C.L., Mahon, A.R., Chadderton, W.L., and Lodge, D.M. 2011. “Sight-unseen” detection of rare aquatic species using environmental DNA. Conserv. Lett. 4: 150-157.

Jerde, C.L., Chadderton, W.L., Mahon, A.R., Renshaw, M.A., Corush, J., Budny, M.L., Mysorekar, S., and Lodge, D.M., 2013. Detection of Asian carp DNA as part of a Great Lakes basin-wide surveillance program. Canadian Journal of Fisheries and Aquatic Sciences 70, 522-526.

Kolar, C.S., Chapman, D.C., Courtenay, W.R., Housel, C.M., Williams, J.D., and Jennings. D.P., 2007. Bigheaded Carps: A Biological Synopsis and Environmental Risk Assessment. American Fisheries Society, Bethesda, MD.

Lawrie AH. 1970. The sea lamprey in the Great Lakes. Transactions of the American Fisheries Society. 99(4): 766-775.

Lodge, D.M., Turner, C.R., Jerde, C.L., Barnes, M.A., Chadderton, L., Egan, S.P., Feder, J.L., Mahon, A.R., and Pfrender, M.E., 2012. Conservation in a cup of water: estimating biodiversity and population abundance from environmental DNA. Molecular Ecology 21, 2555-2558.

MacKenzie, D.I., Nichols, J.D., Sutton, N., Kawanishi, K., and Bailey, L.L. 2005. Improving inferences in populations studies of rare species that are detected imperfectly. Ecology 86(5): 1101-1113.

Minamoto, T., Yamanaka, H., Takahara, T., Honjo, M., Kawabata, Z., 2012. Surveillance of fish species composition using environmental DNA. Limnology 13(2), 193-197.

Ogram, A., Sayler, G.S., and Barkay, T. 1987. The extraction and purification of microbial DNA from sediments. J. Microbiol. Methods 7: 57-66.

Piaggio, A.J., Engeman, R.M., Hopken, M.W., Humphrey, J.S., Keacher, K.L., Bruce, W.E., and Avery, M.L., 2014. Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA. Mol. Ecol. Res. 14: 374-380.

Pilliod, D.S., Goldberg, C.S., Arkle, R.S., and Waits, L.P., 2013. Estimating Occupancy and Abundance of Stream Amphibians Using Environmental DNA from Filtered Water Samples. Canadian Journal of Fisheries and Aquatic Sciences. 70: 1123-1130.

Smith B., Tibbles J. 1980. Sea Lamprey (Petromyzon marinus) in Lakes Huron, Michigan, and Superior: History of Invasion and Control, 1936-1978. Canadian Journal of Fisheries and Aquatic Sciences. 37(11): 1780-1801

Takahara, T., Minamoto, T., Yamanaka, H., Doi, H., and Kawabata, Z.I., 2012. Estimation of Fish Biomass Using Environmental DNA. Plos One 7(4). e35868.

Takahara, T., Minamoto, T., and Doi, H., 2013. Using Environmental DNA to Estimate the Distribution of an Invasive Fish Species in Ponds. Plos One 8(2): e56584.

Thomsen, P.F., Kielgast, J., Iverson, L.L., Wiuf, C., Rasmussen, M., Gilbert, M.T.P., Orlando, L., and Willerslev, E. 2012a. Monitoring endangered freshwater biodiversity using environmental DNA. Mol. Ecol. 21: 2565-2573.

Thomsen, P.F., Kielgast, J., Iversen, L.L., Møller, P.R., Rasmussen, M., and Willerslev, E., 2012b. Detection of a Diverse Marine Fish Fauna Using Environmental DNA from Seawater Samples. PLoS ONE 7(8), e41732.

Valentini, A., Pompanon, F., and Taberlet, P. 2008. DNA barcoding for ecologists. Trends Ecol. Evol. 24(2): 110-117.

 

  • Overview

    Sea lampreys are a species that invaded the Great Lakes presumably following the improvements made to the Welland Canal in 1920. First reported in Lake Erie in 1921, sea lampreys subsequently spread rapidly to the upper Great Lakes and had an established spawning population in all of the upper Great Lakes by 1947 (Applegate 1950). Following their introduction, sea lamprey devastated the commercial and recreational fisheries of the Great Lakes (Lawrie 1970). Sea lamprey populations are currently managed by various control techniques employed by the Great Lakes Fisheries Commission (GLFC). A significant method of control is the use of chemicals to target the larval phase of the lamprey life cycle (Smith and Tibbles 1980). 

    Spawning sea lamprey exhibit potamodromous behavior, leaving the open water of the Great Lakes for tributaries where they swim upstream to spawn. The female lamprey lays between 30,000 to 100,000 eggs into a nest while the male simultaneously fertilizes the eggs. The fertilized eggs hatch into ammocoetes about 18-21 days post fertilization. The ammocoetes spend approximately 4-6 years burrowed in the sediment of the stream where they filter feed on detritus and algae (Applegate 1950). This sedentary lifestyle as a filter feeder makes the ammocoete a prime target for chemical control. However, due to limitations in budget, personnel, and overcoming geographic logistics it is not feasible to treat every sea lamprey-infested stream of the Great Lakes. Therefore, the GLFC must prioritize streams according to estimated lamprey abundance, known treatment costs, and available budget.

    Individual stream treatment needs are currently determined using a variety of assessment techniques which include semi-quantitative surveys of potential habitat as well as sea lamprey ammocoete abundance. These surveys are combined with expert judgement of streams gained by managers through years of sea lamprey control. This information is then used by managers to prioritize streams for treatment. The assessment techniques are presumed to have low precision. The idea behind the assessments is not to obtain actual population densities, but rather to prioritize streams for treatment in order to achieve the maximum return on investment of sea lamprey killed per dollar spent. Ranking some streams for treatment can be difficult due to stream conditions (MacKenzie et al. 2005; Darling and Mahon 2011). Providing a quantitative assessment using eDNA, which is minimally labor intensive, could assist management with stream prioritization by supplementing traditional assessment techniques. 

    Resource agencies have recently adopted molecular surveillance techniques to detect aquatic invasive species (Darling and Mahon, 2011; Lodge et al., 2012; Jerde et al., 2013). One example of a molecular surveillance technique has been the analysis of water samples for the presence of eDNA using polymerase chain reaction (PCR). Recently researchers used eDNA to look for presence or abundance of multi-cellular organisms (Pilliod 2013; Takahara 2012) in water samples. Some aquatic species such as Asian carp are easily detected using eDNA due to their life history, distribution, and DNA shedding characteristics (Kolar et al. 2007; Jerde et al. 2011; Jerde et al. 2013). Conventional polymerase chain reaction (cPCR) techniques target sections of the mitochondrial DNA of the species in question. Conventional and quantitative PCR has been used to monitor for the presence of silver and bighead carp throughout the Chicago Area Waterway System (CAWS) and the Mississippi, Illinois, Wabash and Ohio rivers, and portions of Lake Erie. Detection assays for eDNA are now developed for numerous terrestrial and aquatic species of native or exotic origin (Ficetola et al. 2008; Goldberg et al. 2011; Dejean et al., 2012; Foote et al. 2012; Takahara et al., 2012; Thomsen et al., 2012a; Thomsen et al., 2012b; Egan et al. 2013; Goldberg et al., 2013; Pilliod et al., 2013; Takahara et al. 2013; Piaggio et al. 2014). Environmental DNA is also useful in detecting various species in lotic aquatic environments (Dejean et al. 2011; Jerde et al. 2011; Minamoto et al., 2012; Thomsen et al. 2012a).

    Previous researchers have produced sea lamprey-specific eDNA primers and cPCR assays (Gingera et al., 2016). These assays have been optimized and validated to show they can detect the presence of eDNA from spawning and larval sea lamprey in streams. In addition, they have also found positive correlations with larval densities in a laboratory study. The cPCR assays developed by Gingera et al. are effective at determining presence or absence of the sea lamprey in a stream. However, cPCR analysis of eDNA samples can be challenging due to the presence of non-target DNA and non-specific amplification.  In addition to the challenge of interpreting smeared gels with multiple bands, cPCR analysis requires the additional step of sequencing confirmation.

    An alternative method to cPCR for eDNA detection is quantitative PCR (qPCR, or real-time PCR, probe-based PCR, fluorescence-based PCR). Quantitative PCR assays have primers that bind to the target DNA template and also use a probe that is specific to the DNA sequence of the species of interest. There has been research to suggest that qPCR technologies allow correlation of eDNA quantity to species density and distribution (Takahara et al. 2012; Thomsen et al. 2012a). These types of assays have greater sensitivity as well as increased species specificity. The fluorescence obtained during qPCR analysis is able to provide quantitative estimates of DNA in a sample without the need for additional gel analysis and genetic sequencing.
    The main objective of this research is to determine if sea lamprey eDNA levels correlate with larval and adult abundance. First, we will conduct density trials with known numbers of sea lamprey under highly controlled laboratory settings. Then, we will collect and analyze water samples from actual streams with different densities of lamprey to demonstrate the utility of this tool under more natural settings. In addition to helping managers rank streams for treatment, this information could also be used to estimate the effectiveness of barriers and traps, and provide quantification of adult spawning runs in streams that are not currently trapped. Validated qPCR markers will be used to test detection probabilities and assess our ability to estimate adult and larval sea lamprey abundance in water collected from tanks and streams that contain sea lamprey.

    Objective

    1. The goal of this project is to determine if eDNA abundance is be related to lamprey abundance. We will specifically test two hypotheses:

      Hypothesis 1: If adult sea lamprey shed similar amounts of DNA into the environment, then a system with a greater number of sea lamprey will contain great numbers of sea lamprey eDNA.

      Hypothesis 2: If larval sea lamprey shed similar amounts of DNA into the environment, then a system with a greater number of sea lamprey will contain great numbers of sea lamprey eDNA.
    Technical assistance in support of Sea Lamprey Control Program
    Sources/Usage: Public Domain. View Media Details
    (Public domain.)

     

    References

    Applegate VC. 1950. Natural history of the sea lamprey (Petromyzon marinus) in Michigan. Special scientific report: Fisheries. U.S. Fish and Wildlife Service. 237 pages.

    Darling, J.A., and Mahon, A.R., 2011. From molecules to management: adopting DNA-based methods for monitoring biological invasions in aquatic environments. Environ Res 111: 978-988.

    Dejean, T., Valentini, A., Duparc, A., Pellier-Cuit, S., Pompanon, F., Taberlet, P., and Miaud, C. 2011. Persistence of environmental DNA in freshwater ecosystems. PLoS ONE 6(8): e23398.

    Dejean, T., Valentini, A., Miquel C., Taberlet, P., Bellemain E., and Miaud, C. 2012. Improved detection of an alien invasive species through environmental DNA barcoding: The example of the American bullfrog Lithobates catesbeianus. Journal of Applied Ecology 49: 953-959.

    Egan, S.P., Barnes, M.A., Hwang, C.-T., Mahon, A.R., Feder, J.L., Ruggiero, S.T., Tanner, C.E., and Lodge, D.M. 2013. Rapid invasive species detection by combining environmental DNA with Light Transmission Spectroscopy. Conserv. Lett. 6(6): 402-409.

    Erickson, R.A., Rees, C.B., Coulter, A.A., Merkes, C.M., McCalla, S.G., Touzinsky, K.F., Walleser, L., Goforth, R.R., Amberg, J.J., 2016. Detecting the movement and spawning activity of bigheaded carps with environmental DNA. Molecular Ecology Resources. 2016 May 1

    Ficetola, G.F., Miaud, C., Pompanon, F., and Taberlet, P. 2008. Species detection using environmental DNA from water samples. Biol. Lett. 4: 423-425.

    Foote, A.D., Thomsen, P.F., Sveegaard, S., Wahlberg, M., Kielgast, J., Kyhn, L.A., Salling, A.B., Galatius, A., Orlando, L., and Gilbert, M.T.P. 2012. Investigating the potential use of environmental DNA (eDNA) for genetic monitoring of marine mammals. PLoS ONE 7(8): e41781.

    Gelman A, Hill J. Data analysis using regression and multilevelhierarchical models. Cambridge: Cambridge University Press; 2007.

    Gingera TD, Steeves TB, Boguski DA, Whyard S, Li Weiming, Docker MF. 2016. Detection and identification of lampreys in Great Lakes streams using environmental DNA. Journal of Great Lakes Research. 42: 649-659.

    Goldberg, C.S., Pilliod, D.S., Arkle, R.S., and Waits, L.P. 2011. Molecular detection of vertebrates in stream water: A demonstration using rocky mountain tailed frogs and Idaho giant salamanders. PLoS ONE 6(7): e22746.

    Goldberg, C.S., Sepulveda, A., Ray, A., Baumgardt, J., Waits, L.P., 2013. Environmental DNA as a new method for early detection of New Zealand mudsnails (Potamopyrgus antipodarum). Freshwater Science, 32(3): 792-800.

    Jerde, C.L., Mahon, A.R., Chadderton, W.L., and Lodge, D.M. 2011. “Sight-unseen” detection of rare aquatic species using environmental DNA. Conserv. Lett. 4: 150-157.

    Jerde, C.L., Chadderton, W.L., Mahon, A.R., Renshaw, M.A., Corush, J., Budny, M.L., Mysorekar, S., and Lodge, D.M., 2013. Detection of Asian carp DNA as part of a Great Lakes basin-wide surveillance program. Canadian Journal of Fisheries and Aquatic Sciences 70, 522-526.

    Kolar, C.S., Chapman, D.C., Courtenay, W.R., Housel, C.M., Williams, J.D., and Jennings. D.P., 2007. Bigheaded Carps: A Biological Synopsis and Environmental Risk Assessment. American Fisheries Society, Bethesda, MD.

    Lawrie AH. 1970. The sea lamprey in the Great Lakes. Transactions of the American Fisheries Society. 99(4): 766-775.

    Lodge, D.M., Turner, C.R., Jerde, C.L., Barnes, M.A., Chadderton, L., Egan, S.P., Feder, J.L., Mahon, A.R., and Pfrender, M.E., 2012. Conservation in a cup of water: estimating biodiversity and population abundance from environmental DNA. Molecular Ecology 21, 2555-2558.

    MacKenzie, D.I., Nichols, J.D., Sutton, N., Kawanishi, K., and Bailey, L.L. 2005. Improving inferences in populations studies of rare species that are detected imperfectly. Ecology 86(5): 1101-1113.

    Minamoto, T., Yamanaka, H., Takahara, T., Honjo, M., Kawabata, Z., 2012. Surveillance of fish species composition using environmental DNA. Limnology 13(2), 193-197.

    Ogram, A., Sayler, G.S., and Barkay, T. 1987. The extraction and purification of microbial DNA from sediments. J. Microbiol. Methods 7: 57-66.

    Piaggio, A.J., Engeman, R.M., Hopken, M.W., Humphrey, J.S., Keacher, K.L., Bruce, W.E., and Avery, M.L., 2014. Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA. Mol. Ecol. Res. 14: 374-380.

    Pilliod, D.S., Goldberg, C.S., Arkle, R.S., and Waits, L.P., 2013. Estimating Occupancy and Abundance of Stream Amphibians Using Environmental DNA from Filtered Water Samples. Canadian Journal of Fisheries and Aquatic Sciences. 70: 1123-1130.

    Smith B., Tibbles J. 1980. Sea Lamprey (Petromyzon marinus) in Lakes Huron, Michigan, and Superior: History of Invasion and Control, 1936-1978. Canadian Journal of Fisheries and Aquatic Sciences. 37(11): 1780-1801

    Takahara, T., Minamoto, T., Yamanaka, H., Doi, H., and Kawabata, Z.I., 2012. Estimation of Fish Biomass Using Environmental DNA. Plos One 7(4). e35868.

    Takahara, T., Minamoto, T., and Doi, H., 2013. Using Environmental DNA to Estimate the Distribution of an Invasive Fish Species in Ponds. Plos One 8(2): e56584.

    Thomsen, P.F., Kielgast, J., Iverson, L.L., Wiuf, C., Rasmussen, M., Gilbert, M.T.P., Orlando, L., and Willerslev, E. 2012a. Monitoring endangered freshwater biodiversity using environmental DNA. Mol. Ecol. 21: 2565-2573.

    Thomsen, P.F., Kielgast, J., Iversen, L.L., Møller, P.R., Rasmussen, M., and Willerslev, E., 2012b. Detection of a Diverse Marine Fish Fauna Using Environmental DNA from Seawater Samples. PLoS ONE 7(8), e41732.

    Valentini, A., Pompanon, F., and Taberlet, P. 2008. DNA barcoding for ecologists. Trends Ecol. Evol. 24(2): 110-117.