Molecular Tools for Management of Threatened and Endangered Species

Federal Awarding Agency: U.S. Army Corps of Engineers,
Engineer Research and Development Center
2902 Newmark Dr
Champaign, IL 61822

Funding Opportunity No: W81EWF-22-SOI-0009
CFDA No: 12.630
Statutory Authority: 10 USC 2358

Program Title: Molecular Tools for Management of Threatened and Endangered Species
Issue Date: 28 January 2022
Statement of Interest/Qualifications Due Date: 25 February 2022/2:00 PM/CST
Full Application Package Due Date, if Invited: 18 March 2022/2:00 PM/CST
Estimated Award Ceiling: $64,300.00
Expected Number of Awards: 1

 

Section I: Funding Opportunity Description

Background:
The government seeks research and technical support for the development and demonstration of molecular tools to assess at-risk species occurrence, genomics, and disease risk on military installations from which samples were collected (Fort McCoy, WI, and Camp Grayling, MI). Exact analyses conducted and the need for additional sample collection will be decided based upon input from installation, ERDC-CERL, and CESU partners. It is anticipated that results from these analyses will be relevant for management throughout the focal species ranges. Details of primary tasks are as follows:

Task 1: DNA metabarcoding to assess freshwater aquatic communities
Fort McCoy, WI comprises 4,400 acres of wetlands (NRCT, 2015) that harbor diverse communities, including a number of at-risk species such as wood turtle, Glyptemys insculpta, Blanding’s turtle, Emydoidea blandingii, and four amphibian species considered to be either state endangered (i.e., Blanchard’s cricket frog, Acris blanchardi) or species of special concern (i.e., the four-toed salamander, Hemidactylium scutatum; the pickerel frog, Lithobates palustris; and the northern leopard frog, Lithobates pipiens). In addition, Wisconsin Department of Natural Resources currently regulates 99 aquatic invasive species (64 animals and 35 plants) that pose a risk to Fort McCoy’s freshwater ecosystems. Understanding where these at-risk and invasive species occur across the installation is a first step in determining effective management strategies. Ponds, ephemeral pools, and streams throughout Fort McCoy will be sampled at three time periods (spring, summer, and fall) during 2022. Specific sampling locations and timing will be determined in consultation with CERL and Fort McCoy Natural Resources Branch staff, based on the natural history of focal species, historical records, and availability of suitable habitat. Sampling estimates include 10 – 30 locations per sampling period with three replicate water samples collected in 1-liter bottles at each location and stored on ice. Sampling effort will include a 1-liter bottle of molecular-grade water at each site to serve as a control. Water samples will be filtered using a vacuum pump through 0.80 μm cellulose nitrate filter. Filters will be stored in vials of cetyl trimethylammonium bromide (CTAB) buffer until DNA extraction.

DNA will be extracted from sample filters using a modified phenol–chloroform–isoamyl alcohol extraction (Renshaw et al., 2015)— a commonly used method to isolate DNA from substrates containing high levels of PCR inhibitors (e.g., humic substances), which can inhibit downstream applications (Alaeddini, 2012; Turner et al., 2014; Eichmiller et al., 2015). Extracted DNA will be quantified via Qubit fluorometry before generating community metabarcode data via a multi-primer/locus approach (e.g., Evans et al., 2015; Corse et al., 2019). Vertebrate, plant and invertebrate primer sets will be used to generate sequence reads for each eDNA sample collected.

Task 2: DNA analyses of bacterial pathogens of ticks to assess disease risk.
Ticks are renowned as vectors of disease-causing agents to humans and responsible for nearly 95% of vector-borne diseases in the United States (Eisen et al. 2017). In particular, Lyme disease, caused by the spirochete pathogen, Borrelia burgdorferi s.s., can result in devastating human health consequences. Soldiers involved in training exercises, as well as installation natural resources personnel, can be particularly at risk due to their increased contact with vectors encountered on military ranges (Garcia et al. 2017). Tick species will be sampled on Fort McCoy, WI, via cloth dragging and CO2 trap cloths. Sample sites will be determined based on habitat suitability for tick species and input from CERL and Fort McCoy personnel. At each site, five standard 150-m transects will be established in suitable tick habitat (e.g., leaf litter or grass present in deciduous/mixed forest or ecotonal edge between forest and grassland with evidence of animal activity). These transects will be revisited seasonally (spring, summer, fall) with specific timing dependent on weather and access.

A subsample of 50 ticks will be included in pathogen analyses. Pathogen diagnostics will be performed via DNA extraction and amplification using standard RT-PCR methods in a BSL-2/ACL-3 lab. Ixodes scapularis samples will be tested using a CDC screening algorithm for Anaplasma phagocytophilum, Babesia microti, Borrelia burgdorferi s.s., Borrelia miyamotoi, and Ehrlichia muris euclairensis.

Task 3: DNA metabarcoding to identify plant-pollinator interactions.
Pollinator biomonitoring is critical to both assess the status and trend of at-risk pollinators and evaluate the impacts of biodiversity loss on ecosystem services. Fort McCoy, WI, houses two federally endangered pollinators, the Karner Blue butterfly (Lycaedis melissa samuelis) and the Rusty Patched Bumblebee (Bombus afinis). Fort McCoy is also home to several other pollinator species of conservation concern including the Ottoe Skipper (Hesperia ottoe; state endangered), Regal Fritillary (Speyeria idalia; state endangered), Frosted Elfin butterfly (Callophrys irus; state threatened), Dusted Skipper (Atrytonopsis hianna; special concern) and Leonard’s Skipper (Hesperia leonardus; special concern). Rapid, efficient, and accurate assessment of pollinator communities is a conservation imperative to inform adaptive management strategies and stanch the loss of this critical component of biodiversity.

For this task, traditional pollinator survey methods will be compared to eDNA metabarcoding of flowers to assess effectiveness of eDNA metabarcoding for surveying for at-risk pollinator species and identifying plant-pollinator interactions. Initial focus will be on two pollinator species that are petitioned for federal listing under the Endangered Species Act, the Frosted Elfin and the Regal Fritillary. Traditional surveys will be conducted for both species at Fort McCoy, WI. Survey plots will be determined in collaboration with CERL and Fort McCoy personnel and based on presence of host plants, previous survey efforts, and access. Plot size will vary based on abundance and distribution of host plants. Within plots, surveys will be conducted both via random walk, focusing on presence of host plants, and via fixed 110 x 10 m transects. Surveys will be conducted throughout the active flight period (approximately April–June) with each plot and transect surveyed at least three times. Each transect walkthrough will occur at a steady pace, start/stop times recorded, and host plant and pollinator species observations tallied. Weather conditions and other environmental data at each patch will be measured and documented prior to counts. Counts will only be conducted if weather is within the range of optimal conditions as specified by U.S. Fish and Wildlife Service (2019). If no (or few) butterflies are detected during walkthrough, eggs, larvae, and evidence of larval activity on host plants (e.g., feeding damage) will be documented.

To assess effectiveness of eDNA metabarcoding for assessing pollinators, target flowers will be randomly chosen within survey areas (above). Target flowers will be identified to species, collected, and preserved in ATL buffer. Trace pollinator eDNA left on the flowers will be extracted using a modified Qiagen DNeasy protocol. Extracted eDNA from flowers will then be subjected to metabarcoding. Once PCR and library preparation have been completed, samples will be submitted to the UIUC Keck Core Sequencing Facility for sequencing on Illumina Platforms. Illumina data will be converted from raw sequences to taxonomic assignments using a custom pipeline for reproducible analysis of metabarcoding data: metaBEAT v0.97.78. Using these data, species will be catalogued via conventional observations as well as eDNA metabarcoding.

Task 4: Genomic analyses for at-risk bats (Myotis spp.).
Several species of bat across the US have experienced drastic declines since 2006, primarily as a result of disease (white-nose syndrome, WNS), with some species reduced by over of 90% of their pre-WNS numbers. Where populations persist, survivors may provide clues to disease resistance, with recent research finding genetic differences between bats killed by white-nose-syndrome and those that survived (Gignoux-Wolfsohn et al. 2021). At-risk bats (Myotis sp.) will be sampled at Camp Grayling, MI, and tissue samples collected via wing biopsy punch for genomic analysis. Data for at-risk bats are particularly lacking for the northern Midwest region. These data will help fill that gap and enable comparison to studies from other regions for range-wide assessment. Work accomplished under this task will include sample collection, laboratory analyses, and summarization. Data will be used to populate reports to military installations regarding the statuses of at-risk species on their properties.

Public Benefit:
The benefits of biodiversity to public good have long been recognized. This work will aid in the maintenance of biodiversity by assisting in natural resource management efforts and conservation of endangered and at-risk wildlife species. The public will benefit from this work through enhanced wildlife biodiversity, both on Army installations and on nearby public and private lands. Designation of a species as federally threatened or endangered by the U.S. Fish and Wildlife Service recognizes that a species is a nationally important component of biodiversity in the United States and that federal agencies must support as necessary conservation and recovery objectives for listed species.

Section II: Award Information

Responses to this Request for Statements of Interest will be used to identify potential investigators for studies to be sponsored by the Engineer Research and Development Center to provide research support in the form of molecular analyses—namely, DNA metabarcoding, PCR assay, and genomic analysis—for management of threatened and endangered species. The estimated level of funding for FY22 is approximately $64,126.

Government Involvement:
Substantial government involvement is anticipated for this project. The Government’s technical contributions include:

1. Participation in study design and execution.
2. Expertise in military installation natural resource management
3. Coordination between installation personnel and university researchers

Section III: Eligibility Information

1. Eligible Applicants – This opportunity is restricted to non-federal partners of the Great River and Great Lakes – Northern Forest Cooperative Ecosystems Studies Unit (CESU).
2. Cost Sharing – This action will be 100% funded by USACE.

Section IV: Application and Submission Information – Two Phase Process

Phase I: Submission of a Statement of Interest/Qualifications.

1. Materials Requested for Statement of Interest/Qualifications:
   • Please provide the following via e-mail attachment to: [email protected]
   • (Maximum length: 2 pages, single-spaced 12 pt. font).
     • Name, Organization and Contact Information
     • Brief Statement of Qualifications (including):
       • Biographical Sketch,
       • Relevant past projects and clients with brief descriptions of these projects,
       • Staff, faculty or students available to work on this project and their areas of expertise,
       • Any brief description of capabilities to successfully complete the project you may wish to add (e.g. equipment, laboratory facilities, greenhouse facilities, field facilities, etc.).
   • A proposed budget is NOT requested at this time.
   • The administrative point of contact is Stacy Thurman; [email protected]
2. Statement of Interest/Qualifications shall be submitted NO LATER THAN 25 February 2022/2:00 PM/CST Based on a review of the Statements of Interest received, an investigator or investigators will be invited to move to Phase II which is to prepare a full study proposal. Statements will be evaluated based on the investigator’s specific experience and capabilities in areas related to the study requirements.

Phase II: Submission of a complete application package to include a full technical proposal including budget, if invited.

1. Address to Request Application Package. The complete funding opportunity announcement, application forms, and instructions are available for download at Grants.gov. The administrative point of contact is Stacy Thurman;  [email protected]
2. Content and Form of Application Submission. All mandatory forms and any applicable optional forms must be completed in accordance with the instructions on the forms and the additional instructions below.
   • SF 424 R&R - Application for Federal Assistance
   • Full Technical Proposal – Discussion of the nature and scope of the research and technical approach. Additional information on prior work in this area, descriptions of available equipment, data and facilities, and resumes of personnel who will be participating in this effort should also be included.
   • Cost Proposal/Budget – Clear, concise, and accurate cost proposals reflect the offeror’s financial plan for accomplishing the effort contained in the technical proposal. As part of its cost proposal, the offeror shall submit cost element breakdowns in sufficient detail so that a reasonableness determination can be made. The SF 424 Research & Related Budget Form can be used as a guide. The cost breakdown should include the following, if applicable:
     • Direct Labor: Direct labor should be detailed by level of effort (i.e. numbers of hours, etc.) of each labor category and the applicable labor rate. The source of labor rates shall be identified and verified. If rates are estimated, please provide the historical based used and clearly identify all escalation applied to derive the proposed rates.
     • Fringe Benefit Rates: The source of fringe benefit rate shall be identified and verified.
     • Travel: Travel costs must include a purpose and breakdown per trip to include destination, number of travelers, and duration.
     • Materials/Equipment: List all material/equipment items by type and kind with associated costs and advise if the costs are based on vendor quotes and/or engineering estimates; provide copies of vendor quotes and/or catalog pricing data.
     • Subrecipient costs: Submit all subrecipient proposals and analyses. Provide the method of selection used to determine the subrecipient.
     • Tuition: Provide details and verification for any tuition amounts proposed.
     • Indirect Costs: Currently the negotiated indirect rate for awards through the CESU is 17.5%.
     • Any other proposed costs: The source should be identified and verified.
3. Application package shall be submitted NO LATER THAN 18 March 2022/2:00 PM/CST
4. Submission Instructions: Applications may be submitted via e-mail to [email protected] Format all documents to print on Letter (8 ½ x 11”) paper.

Section V: Application Review Information

1. Peer or Scientific Review Criteria: In accordance with DoDGARs 22.315(c), an impartial peer review will be conducted. Subject to funding availability, all proposals will be reviewed using the criteria listed below (technical and cost/price). All proposals will be evaluated under the following two criteria which are of descending importance.
   • Technical (items i. and ii. are of equal importance):
     • Technical merits of proposed R&D.
     • Potential relationship of proposed R&D to DoD missions.
   • Cost/Price: Overall realism of the proposed costs will be evaluated.
2. Review and Selection Process
   • Categories: Based on the Peer or Scientific Review, proposals will be categorized as Selectable or Not Selectable (see definitions below). The selection of the source for award will be based on the Peer or Scientific Review, as well as importance to agency programs and funding availability.
     • Selectable: Proposals are recommended for acceptance if sufficient funding is available.
     • Not Selectable: Even if sufficient funding existed, the proposal should not be funded.
3. The Government reserves the right to award some, all, or none of proposals. When the Government elects to award only a part of a proposal, the selected part may be categorized as Selectable, though the proposal as a whole may not merit such a categorization.
   • No other criteria will be used.
   • Prior to award of a potentially successful offer, the Grants Officer will make a determination regarding price reasonableness.

Section VI: Award Administration Information

1. Award Notices. Written notice of award will be given in conjunction with issuance of a cooperative agreement signed by a Grants Officer. The cooperative agreement will contain the effective date of the agreement, the period of performance, funding information, and all terms and conditions. The recipient is required to sign and return the document before work under the agreement commences. Work described in this announcement SHALL NOT begin without prior authorization from a Grants Officer.
2. Administrative Requirements. The cooperative agreement issued as a result of this announcement is subject to the administrative requirements in 2 CFR Subtitle A; 2 CFR  Subtitle B, Ch. XI, Part 1103; and 32 CFR Subchapter C, except Parts 32 and 33.
3. Reporting. See 2 CFR Sections 200.327 for financial reporting requirements, 200.328 for performance reporting requirements, and 200.329 for real property reporting requirements.

Section VII: Agency Contact

Stacy Thurman, Grants Specialist
US Army Corps of Engineers, Engineer Research and Development Center
3909 Halls Ferry Road
Vicksburg, MS 39180-6199
[email protected]