The objective of this project is to demonstrate and validate a rapid approach for simultaneously and cost-effectively procuring information on multiple critical aspects of bat populations using DNA from non-invasively collected guano pellets. The approach, referred to as multifaceted DNA metabarcoding (MDM), is an innovative combination of next-generation sequencing (NGS), DNA metabarcoding, and molecular scatology. Several federally protected bat species occur on Department of Defense (DoD) lands and, since the 1990s, DoD has expended more than $20 million on just two bat species: the endangered gray bat (Myotis grisescens) and Indiana bat (M. sodalis). It is expected that white-nose syndrome (WNS), which is spreading across the continent and decimating bat populations, will not only negatively impact these two species but will also result in the federal listing of additional bat species occurring on DoD lands. Several different types of data are critical for effectively managing threatened, endangered, and at-risk (TES-AR) bats, but a variety of conventional field and lab studies are required to obtain these diverse data, and each requires significant time and expense. The MDM approach provides many of the same types of data as conventional approaches, but with improvements in speed, ease of sample collection, and relative impacts on the bats. Specific objectives of this project are to: (1) validate the accuracy of this approach for quantifying multiple types of population parameters; (2) carry out a full-scale demonstration of this approach on two problematic populations of bats; and (3) compare the cost, effort, and accuracy of this approach against those required for conventional approaches.

DNA barcodes are gene sequences unique to each species or group of species that can be used to identify samples of unknown origin. Metabarcoding refers to the practice of using DNA barcodes to characterize the species contributing to a sample with a mixed DNA pool (e.g., soil, feces). In MDM, DNA from each sample is enriched for select genetic markers and assigned a unique oligonucleotide tag, after which samples are pooled and assayed simultaneously in an NGS run. Multiple genetic markers are used to provide information on each sample, including species identification, sex determination, presence/absence of WNS, and individual DNA genotypes (or fingerprinting) that can be used for population size estimates. In addition, the team will use DNA metabarcoding, which is an emerging capability that employs next-generation DNA sequencing to characterize the diversity of organisms from a particular taxonomic group and quantify the diversity of diet items and parasites. The vast data-generative power of NGS platforms allows for a single sequencing run to provide data on multiple different DNA marker types from multiple focal groups for large numbers of samples. This approach is of particular value when taxa are difficult to directly observe, sample, or count, are difficult to handle without risking significant injury to the animal, or that occur in large populations. Bats are a classic example of such taxa. MDM should provide an unprecedented scope of data about bat populations by simply collecting guano pellets in roost areas or along flight paths.

Benefits of MDM include: (1) enhanced WNS surveillance, (2) identification of critical foraging resources used by bats, and (3) ability to track changes in bat health parameters (i.e., changing population size or parasite loads). It will also allow for rapid determination of the species utilizing different roost sites and the size and makeup of the colony. Several TES-AR bats inhabit DoD lands, with additional species likely to be listed in the near future due to WNS. Critical data for bat monitoring and management are often difficult to obtain without extensive fieldwork, cost, and stress to the bats. The MDM approach should provide these data with little or no stress to bats. Furthermore, the team expects that this approach will be as accurate, more informative, and more cost- and labor-efficient than the combined efforts of multiple studies that would be required to obtain similar data using conventional approaches. This project will demonstrate and validate the accuracy and efficiency of MDM so that it gains acceptance for widespread use in quantifying critical parameters in bat populations on DoD lands. Finally, MDM can be applied to any organism for which individual-level genetic material can be procured. (Anticipated Project Completion - 2018)