Noninvasive Multifaceted DNA Metabarcoding for Characterizing TES Bats

R. Lance, US Army ERDC Environmental Laboratory, Vicksburg, MSE. Britzke, US Army ERDC Environmental Laboratory, Vicksburg, MSC. Edwards, Missouri Botanical Gardens, St. Louis, MO

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

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

Noninvasive Multifaceted DNA Metabarcoding

Molecular Scatology

NGS• Single upscale

run can produce 800,000 sequences

• Single-molecule sensitivity

• 1 GS Mini Jr run ≈ 90,000 sequences for about $1,000

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 MARKERMID 1 MID 2

GENDER MARKERMID 1 MID 2

SPECIES MARKERMID 1 MID 3

GENDER MARKERMID 1 MID 3

SPECIES MARKER MID 3MID 2

MID 3MID 2 GENDER MARKER

Scat 1 {Scat 2 {Scat 3 {

Basic Study Plan

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

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

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

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.

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

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

Tech Transfer/Acceptance• Robyn Niven, USFWS Liaison• 2017 Webinar for DoD user community and

regulatory community• 2017 How-To & Best Practices Technical Note

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

Qiagen Dneasy 0

CTAB Qiagen Dneasy 0.5

CTAB 0.5 Qiagen Mericon

Qiagen QIAamp

Stool

0

2

4

6

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10

Successful Species Barcode PCRs by DNA Isolation Protocol

SucceededFailed

Isolation Protocol0 = Recommended Elution, 0.5 = 2X Dilute Elution

Guan

o Pe

llet S

ampl

es

Questions?