1. Noninvasive Multifaceted DNA Metabarcoding for Characterizing TES Bats R. Lance, US Army ERDC Environmental Laboratory, Vicksburg, MS E. Britzke, US Army ERDC Environmental Laboratory, Vicksburg, MS C. Edwards, Missouri Botanical Gardens, St. Louis, MO

2. 4 (going on 5) TES Bats on Installations Gray Bat (Myotis grisescens) Indiana Bat (M. sodalis) Hawaiian Hoary Bat (Lasiurus cinereus) Lesser Long- nosed Bat (Leptonycteris yerbabuenae) Northern Long-eared Bat (M. septentrionalis) Background

3. Maximize our ability to obtain data from bats in noninvasive manner Minimize stressors Minimize cost More data per unit effort Get data that might otherwise be unavailable Genetic material contained in guano pellets (or scat, in general) can provide a wealth of data. Unique DNA sequences (e.g. barcodes) can identify species (e.g. bats, diet items, parasites) and gender. Others can be used as individual-level tags or genotypes for capture-recapture population estimates. Next Generation Sequencing (NGS) allows us to deal with MANY samples and MANY markers simultaneously. How do we combine molecular scatology, DNA barcoding, and NGS capabilities to maximize noninvasive data procurement for bats? Background

4. Noninvasive Multifaceted DNA Metabarcoding

5. Molecular Scatology

6. NGS Single upscale run can produce 800,000 sequences Single-molecule sensitivity 1 GS Mini Jr run ≈ 9 0,000 sequences for about $1,000

7. Metabarcoding Multiplex Identifier Adaptors (MIDS) provide unique code for each sample e.g., combos of 25 MIDs = 600 unique barcodes = 600 samples At least 140 “official” MIDS available SPECIES MARKER MID 1 MID 2 GENDER MARKER MID 1 MID 2 SPECIES MARKER MID 1 MID 3 GENDER MARKER MID 1 MID 3 SPECIES MARKER MID 3 MID 2 MID 3 MID 2 GENDER MARKER Scat 1 { Scat 2 { Scat 3 {

8. Basic Study Plan

9. Demo 1: Fort Drum Ryan von Linden Karl Butchkoski Fort Drum: MDM demonstration incorporating determinations/estimates of -- Species Gender Population size WNS levels Parasites Diet Roger W. Barbour

10. Study Design: Collect 500-600 guano pellets from under roost during pre-parturition maternity season and then again during weaning period. Pellets subjected to MDM. Execute parallel conventional data collection for results and costs comparisons

11. Fort Huachuca: MDM demonstration incorporating determinations/estimates of -- Species Gender Population size Parasites Diet Roger W. Barbour www.mnh.si.edu Demo 2: Fort Huachuca

12. Study Design: Collect 1000-1500 guano pellets from roost cave in early summer when agave scarce and again during early autumn during weaning period and agave scarce again. Sacrifice small number of cave myotis for parasite estimate comparisons Same process for both species.

13. Relative Pros:Cons – Conventional Approaches Conventional approaches require capture and handling Risks of injury and roost abandonment Risks of disease spread Require permit Some species easier to capture than others Can observe degree of WNS Determination of parasite loads generally require sacrificing bat Not likely for TES or WNS susceptible bats Diet studies requires entomological expertise and considerable time Generally accurate to level of order or family Costs ≈ $70,000 for very limited effort in obtaining the 6 data classes

14. Relative Pros:Cons – Multifaceted DNA Metabarcoding MDM poses NO risk of bat injury and roost abandonment Permits typically not required Risk of disease spread greatly minimized Cryptic species can be discerned Parasite characterization does not require bat mortality or handling Depending on existing database or data requirements, diet characterization to the level of species Level of individual WNS infection may not be discernible Cost ≈ $20,000 ~ 40% of costs are for initial investment in primers, which may be used multiple times

15. Tech Transfer/Acceptance Robyn Niven, USFWS Liaison 2017 Webinar for DoD user community and regulatory community 2017 How-To & Best Practices Technical Note

16. Aim to demonstrate highly time- and cost-efficient, and accurate, method for obtaining large quantities of data on bats using noninvasive samples Constantly evolving genotyping capabilities

18. Questions?