Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Veterinarian Microbiology & Immunology
Non Technical Summary
Environmental DNA (eDNA) is an emerging technology that indirectly measures the presence of a fish species by detecting its DNA signature in cells shed into water. Although the technique provides qualitative associations it is not quantitative, valid or scientifically defensible when used to monitor species invasion. Nevertheless, questionable data documenting the invasion of Asian carp into the Great Lakes (Jerde et.al, 2011, Conservation Letters 00,1-8) is the basis of numerous lawsuits from Michigan, Wisconsin, Ohio, Minnesota and Pennsylvania against the US Army Corps of Engineers. Similarly, New York Senators Charles Schumer and Kirsten Gillibrand recent request for the Army Corps of Engineers to close the Chicago waterway system to prevent Asian Carp invasion emphasizes the importance of this line of investigation not only to New York but also to the overall health of Great Lakes fisheries. The International Joint Commission (IJC) strongly supports the development of eDNA technology to investigate entry and spread of invasive species. (http://schumer.senate.gov/Newsroom/record.cfm?id=334868). Validated and quantitative eDNA technology will be a broad reaching methodology that is readily adaptable to any fish species whether targeting invasive species or monitoring levels of an endangered or recovering species. Extending eDNA technology will impact many Great Lakes interests especially the Lake Sturgeon Rehabilitation status assessment and provide methods to generate data in support of Ecosystem Modeling and aspects of Fish Recruitment. Our approach is aimed at detecting fish specific eDNA in water samples by employing qPCR methods to provide an exact measure of species presence and density. Validation of eDNA technology will require experimental proof that species density relates to eDNA signal. This aspect of the research will be done in laboratory-controlled conditions in aquaria. The mitochondrial DNA (mtDNA) cytochrome oxidase 1(COI) gene in eDNA samples will be the target to determine the presence and level of invasive species and also define the community composition of fish species at a given locale. We propose to: 1. Establish eDNA the sensitivity and specificity of qPCR assays targeting for bighead, silver, grass and black carp. Parameters of fish density, eDNA stability will be done in laboratory aquaria. 2. Implement eDNA testing at endemic and non-endemic sites in Chicago river estuaries and surrounding Lake Michigan waters. 3. Host two workshops with collaborators and others once we can reveal our new technology and its application to changing biotic conditions.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
With climate change, there are many accounts of new organisms approaching New York waters from the warmer Great Lakes. Anticipating a new wave of aquatic invaders with warming waters, we propose to: 1. Apply a new genomic detection method developed in our laboratory for invading Asian carp and establish detection sensitivity in a controlled environment to provide quantitative abundance predictions; 2. Demonstrate field application for detecting bighead, grass, black and silver Asian carp species into New York; 3. Host a workshop with collaborators and others once we can reveal our new technology and its application to changing biotic conditions. Our major accomplishment will be developing and demonstrating detection of DNA for Asian carp species. This will be a new and scientifically refined environmental technology that can address threats by invading species now permitted by climate change. The test organisms, Asian carp, are major threats to New York waterways. Major impacts from the colonization of New York by these species include: (1) the detrimental effects on native species, especially benthic species; (2) their ability to persist and proliferate in a wide range of habitats; (3) the potential source of contaminant transfer through the food web; and (4) their ability to harbor virus pathogens (Spring of Viremia Carp) that kill other fish in large numbers. The milestones for year 1 include developing additional qPCR sets for grass carp and black carp and set up mesocosms for grass carp. From the middle of year one and through year 2 we will be collecting water samples for eDNA analysis. The intention is to do three collections on different dates at non-endemic sites. One collection from endemic sites should suffice to obtain enough data to make an estimate of Asian carp abundance. Two workshops are planned. The first will likely be held in early spring 2013 and the second 2014. This is enough time to gather data on eDNA sampling to have an impact and demonstrate the utility of eDNA technology. Our major accomplishment will be developing and demonstrating detection of DNA for Asian carp species. This will be a new and scientifically refined environmental technology that can address threats by invading species now permitted by climate change.
Project Methods
Our genomic analysis capability with quantitative PCR (qPCR) that was initially developed for viral and bacterial pathogens can be extended to detect species presence and abundance using DNA from cells shed in water. We will use qPCR to detect DNA from shed cells of specific species in water samples. The steps to develop this capability include defining primers and probes for the object species and common indigenous species (control) targeting the mitochondria cytochrome oxidase 1 gene (CO1) DNA. Since the submission of the pre-proposal we have advanced the technical aspects of qPCR probe and primer design such that we now have a pantropic set of reagents that will simultaneously detect all four species of Asian Carp. Similarly, we have developed a digital subtraction system to select against cross amplification of Great Lakes resident fish species and optimize the pantropic reagents for specificity using barcoded sequences for the CO1 gene. Briefly, in 2003, Paul Hebert proposed DNA barcoding as a way to identify species. Barcoding uses the sequence from a 648-base-pair region in the mitochondrial cytochrome c oxidase 1 gene (CO1) the way a supermarket scanner distinguishes products using the black stripes of the Universal Product Code. Mitochondrial DNA evolves much faster than does nuclear DNA, so each barcode is distinct for an animal species and has a signature that unambiguously assigns it to a position in taxonomic classification. We began this investigation understanding that the major challenge of all qPCR approaches is to develop primers and probes that would not cross-amplify other fish species present in the Great Lakes and would amplify the invasive species under consideration (specificity). From published records at least 160 different fish species inhabit the Great Lakes. Fortunately, most Great Lakes fish have been barcoded such that sequence analysis of individual fish barcodes (sequence mismatch) can be used to predict specificity. With this information we can make an rational decision in selecting primer probe sets for maximal specificity. Sampling will be segregated into endemic and non-endemic sites and collected at different times to avoid the possibility of contamination. Endemic sites will include the lower Illinois River, below the Calumet Sag channel and the electric barriers. This is the site where Asian carp have been detected. eDNA samples will also be obtained from Lake Chicot AR courtesy of Dr Andy Goodwin. This site has a large population of bigheads and will serve as a positive control and an abundance standard. Non-endemic sites will include the Chicago waterfront where eDNA is being tested using the less sensitive techniques developed by Jerde and colleagues. Specifically, water samples will be collected at Lake Calumet and the junction of the Calumet River and Lake Michigan at Calumet Park. Similarly, water samples will be collected from the Chicago River at Dusable Park and further north at Gillston Park in Evanston, Ill. The sites proposed here will be re-sampled and expanded upon depending on our first results.