1. eDNA Analysis and Refinement FY14 GLRI Proposal Overview ACRCC 24 April 2014 US Fish and Wildlife Service MIDWEST REGION US Army Corps of Engineers PLANNING SMART BUILDING STRONG ®

2. USFWS Monitoring and Response Team Support in CAWS Long‐term monitoring and rapid response activities regarding Asian carp throughout the CAWS, above and below the Electric Barrier System. Enhanced sampling with conventional (electrofishing, netting, sidescan sonar, hydroacoustics, rotenone) and novel gears (eDNA, DIDSON, Paupier net) used to document Asian carp population dynamics within the canal system and connecting waterways, provide data for modeling potential population movements (range expansion), document fish behavior in and around the barriers, and determine life stages of Asian carp potentially present. Response activities may be implemented where specific evidence indicates presence of Asian carp above the electric barriers, or if a catastrophic event necessitates immediate action. Outcomes: Continued development of ACRCC’s MRP, as needed to monitor the leading edge of the Asian carp expansion Support of Incident Command System (ICS) response operations as needed Provision of staff, equipment, supplies, and ICS team members as needed. Base Expected FundingGLRI Funding Requested $300,000$520,000

3. USFWS Fisheries Program Capacity for eDNA Sampling and Early Detection In FY13, the Service’s Midwest Fisheries Center, Whitney Genetics Lab began to implement a comprehensive, effective, and efficient program in the Great Lakes to detect incipient invasions. This task will provide USFWS Fish and Wildlife Conservation Office facilities with resources and expertise to conduct integrated, long-term early detection activities in areas outside of the CAWS using eDNA. Funding received under this action item will support water sample collection around the Great Lakes Basin, and samples will be analyzed for eDNA at the Midwest Fisheries Center, Whitney Genetics Lab. Outcomes: Continued eDNA sampling in areas of concern by USFWS Fish and Wildlife Conservation Offices, conducted in close coordination with partners. Continued updating of the Quality Assurance Project Plan to include any necessary updates for collection, handling, and processing of water samples. Base Funding ExpectedGLRI Funding Requested $400,000$650,000

4. USFWS Midwest Region Fisheries Program Capacity for eDNA Sample Processing and Technique Refinement Processing of eDNA samples for Asian carps at the Service’s Midwest Fisheries Center, Whitney Genetics Lab of at least 500 water samples per week from the CAWS and other Great Lakes hotspots. Use of eDNA as a monitoring tool for Asian carps and other AIS is still in early development stages, and is rapidly being improved through research efforts within federal agencies and academic institutions. In order to implement new techniques and methods as they are published, new methods and techniques must be tested and validated to be included in the QAPP, and then implemented in official monitoring programs. Adaptations from the methods must be researched and then validated in at least three different labs. Furthermore, as the Great Lakes monitoring program commences, and additional monitoring sites are added, the lab will be required to increase capacity and efficiency. Higher throughput can be realized with modifications to current procedures and methods, all of which require testing and validation in three labs in order to be adopted into the QAPP. The base funds cover processing of routine monitoring samples up to 500 samples per week. Additional GLRI funds are needed to cover costs of the validation and implementation of new technologies transferred to the monitoring program from the ECALs research. Outcomes: USFWS eDNA sample processing and analysis at a rate of at least 500 samples per week at the Whitney Genetics Lab. Base Funding ExpectedGLRI Funding Requested $900,000

5. Using eDNA as a Stock Assessment Tool to Better Understanding Carp Populations and for Tracking Impacts of Control Measures Use basic eDNA approach as a stock assessment tool that provides more than an early detection assay for invasive carp. Use existing eDNA sampling to provide information on carp populations that otherwise are unattainable without significant “hands on” efforts to capture and process fish. qPCR and next generation sequencing to detect and characterize markers that are only found in one or a few locations: sex-linked markers for determining population sex ratios, mitochondrial and microsatellite markers that provide diversity indices that may be used to cross-validate other methods for estimating population sizes, and telomeric markers that potentially reflect fish age, stress, or health. Be able to discern population characteristics such as sex ratios, population estimate cross-validation, and age ratios or general health indices. Procedures can be adopted by the USFWS Whitney Genetics Laboratory and other labs, and these helpful data can be generated along with more established assays for detection and population estimation of invasive carp. Outcomes: The ability to use existing eDNA sampling methods to provide population-level data on impacts of invasive control efforts. Task/ProductBase FundingGLRI Funding Requested Support for USFWS Monitoring Program $700,000 $125,000 Sex Determination and Detection Trials$100,500 Population Estimate Validation Trials$100,000 Age/Health Determination Trials$60,000 Total$385,500

6. Development of a hand-held, real-time DNA detection kit to detect bigheaded carp DNA to reduce the risk of the transfer of bigheaded carp with baitfish or in other live fish transport Could provide use for law enforcement personnel working in time-sensitive settings; could simplify inspection process Laboratory-based methods still an effective and efficient method to detect the presence of bighead carp or silver carp eDNA for surveillance and monitoring programs. A field-deployable rapid eDNA detection kit could have many other uses in field operations to quickly assess whether the DNA of bighead carp or silver carp are present (e.g., to guide sample collection for refined laboratory-based monitoring). Lucigen® Corporation is currently combining loop-mediated isothermal amplification (LAMP) with a lateral-flow strip to produce cost-effective hand-held devices for the detection of various types of pathogens in both human and animal clinical settings in as little as 12 minutes with detection capacity reportedly equivalent to laboratory-based PCR systems. This device will make use of several markers presently being developed (for any species) Prototype field-deployable devices could be available in early 2015 The development of a field-deployable, hand-held, real-time DNA detection kit does not negate the need for laboratory- based eDNA monitoring and surveillance efforts. The principal advantage of current and next generation field-deployable, real-time DNA detection kits will be the portability and immediate, near real-time results these kits offer to detect the presence of the DNA of bighead carp or silver carp. Outcomes: A hand-held, real-time DNA detection kit for use by field personnel to detect the DNA of bigheaded carp to reduce the risk of the transfer of bigheaded carp with baitfish or in other live fish transport systems Base Funding Expected GLRI Funding Requested 0$100,000 Lysis buffer Incubate Heat Block