Funding Wizard

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.