The recently identified causative agent of White-Nose Syndrome (WNS), Geomyces destructans, has been responsible for the mortality of an estimated 5.7 million North American bats since its emergence in 2006. A primary focus of the National Response Plan, established by US Fish and Wildlife in 2011, was the identification of biological and chemical control options. In an effort to identify potential biological and chemical control options for WNS, six previously described bacterially produced volatile organic compounds (VOCs) and multiply induced Rhodococcus rhodochrous DAP96253 were screened for anti-Geomyces destructans activity. Geomyces destructans conidia and mycelial plugs were exposed to the VOCs and induced Rhodococcus in a closed air space at 15°C and 4°C and evaluated for inhibition of conidia germination and mycelial extension. Additionally, in situ application methods for induced Rhodococcus, such as fixed cell catalyst and fermentation cell paste in non-growth conditions, were screened with positive results. Rhodococcus was assayed for ex vivo activity via exposure to bat tissue ex-plants inoculated with G. destructans conidia. All VOCs inhibited radial growth of mycelial plugs and growth from conidia at both temperatures, with the greatest effect at low temperature (4°C). Induced Rhodococcus completely inhibited growth from conidia at 15°C and had a strong fungistatic effect at 4°C. Induced Rhodococcus inhibited Geomyces destructans growth from conidia when cultured in a shared air space with bat tissue explants inoculated with Geomyces destructans conidia. During the evaluation diffusible brown pigment was observed in G. destructans cultures exposed to induced Rhodococcus or select VOCs. The pigment was induced by light and oxidative challenge and hypothesized to be melanin. Traditional microbiological methods, as well as copper sulfide-silver staining and ultraviolet-visible spectroscopy, were utilized to confirm this hypothesis. This was a noteworthy result as melanin is a known virulence factor in other pathogenic fungi and may play a significant role in WNS. The identification of bacterially produced VOCs and inducible biological agents with anti-Geomyces destructans activity expands the pool of potential biological and chemical control options for WNS and provides wildlife management personnel with tools to combat this devastating disease.

Emergent epizootics are responsible for dramatic declines in wildlife populations over the past few years. An emerging fungal-borne epizootic, called white-nose syndrome (WNS), is responsible for a catastrophic decline of hibernating bats in North America The fungus, Pseudogymnoascus destructans (Pd), is the causative agent of WNS, but to date, we have limited understanding of how an infection with Pd can lead to mortality in hibernating bats. Evidence suggests dehydration is an important part of the pathogenesis of WNS. Cryan et al. (2010) proposed four possible mechanisms by which infection could lead to dehydration. In this study, I tested one of these hypotheses - Pd infection could cause disruption to passive gas-exchange pathways across the wing membranes, thereby causing a compensatory increase in water-intensive pulmonary respiration. I hypothesized total evaporative water loss would be greater when passive gas-exchange was inhibited, especially at low ambient temperatures. I found that bats did not lose more water when passive gas-exchange was retarded (at least within the resolution of my equipment). This study provides evidence against the proposed proximal mechanism that disruption to passive gas-exchange causes dehydration and ultimately death to WNS-infected bats.

North American bats face multiple threats, prompting an increase in bat research and conservation efforts in recent decades. Researchers often use acoustic monitoring, which entails recording bats? echolocation calls and subsequently identifying them to species, typically using automated software. Chapter 1 describes an acoustic monitoring program at eight U.S. national parks that aims to assess changes in bat populations over time. Data collected in 2016-2017 showed that activity levels of the little brown bat (Myotis lucifigus) decreased significantly while other species remained stable. Little brown bats have undergone similar population declines elsewhere due to the disease white-nose syndrome. Chapter 2 investigates whether different versions of bat call identification software are comparable to each other and how accurate they are. For the two software programs tested, agreement among versions was variable and species-dependent. Furthermore, newer versions were more conservative in assigning identifications, though not, on average, more accurate.

White-Nose Syndrome (WNS; Pseudogymnoascus destructans) is responsible for the regional population collapse of many cave-hibernating bat species, including the northern long-eared bat (Myotis septentrionalis), in eastern United States and Canada. I evaluated roosting behavior, habitat selection, and landscape-scale distribution of roosts of the northern long-eared bat during spring emergence and the early maternity season in Mammoth Cave National Park, Kentucky, USA, from 2015 to 2016. Logistic regression analysis comparing habitat features of roosts with random plots indicated selection of roosts reflected the costs of energetic demands by sex and reproductive status. Relative abundance of local bat species was assessed pre- and post-arrival of WNS in the Park during the summer season, with capture rates observed during the progression of WNS indicating that the fungal disease led to declines in the overall abundance of several bat species on the summer landscape, especially the northern long-eared bat. Distributional trends were quantified using spatial point pattern analysis which indicated that bats had clear roosting patterns associated with landscape level features and habitat resources. Monitoring bat populations regionally and at local scales will be imperative to helping conservation efforts for several bat species most affected by WNS.

Volatile organic compounds (VOCs) are organic chemicals typically characterized as having low molecular weight, low solubility in water, and high vapor pressure. Consequently, they readily evaporate from liquid to the gaseous phase at standard temperature and pressure. VOCs are produced by many microorganisms as a result of both uninduced and induced metabolic pathways. Volatile-based microbial inhibition in environments such as soil is well founded, with numerous antimicrobial VOCs and formulations having been identified. Inhibitory VOCs are of particular interest as microbial control agents, as low concentrations of gaseous VOCs have been observed to elicit significant antimicrobial effects. It is believed that this contact-independent antagonism may present unique advantages over traditional microbial control methods, particularly where contact-dependent treatment methods are either impractical or inconvenient. This method may be of particular benefit for managing infections where disease may become pervasive in the population, such as with white-nose syndrome (WNS) among bats. A list of potential antifungal compounds and formulations was compiled by referencing the scientific literature. Screening of compounds and formulations was conducted through toxicity analyses and antimicrobial susceptibility testing for the in vitro ability of VOCs and formulations to inhibit growth of select pathogenic fungi. A dispersal system was developed that entailed electrical circuit and software engineering as well as quantitative analysis to validate consistent and accurate dispersal of potential treatment compounds and formulations. Successful completion of these goals culminated in exposure trials involving live bats to determine any significant toxicological effects. Ex and in situ treatment trials were conducted to determine efficacy of promoting the reduction of disease severity and increasing survivorship of infected bat populations. The identification of volatile-based inhibitory compounds, in conjunction with a novel method for accurate and automated delivery, could prove a promising treatment and prophylactic in combatting microbial pathogenesis and contamination.

No description available.

The Indiana Bat (Myotis sodalis) population had decreased by 56% between 1967 and 2006. In summer 2006, a mysterious disease called White Nose Syndrome was first identified. Since then, the disease killed almost one million bats in North America. Many Biologists believe that both the population decrease before the appearance of the disease and WNS are associated with climate. In a joined effort with Yellowstone Ecological Research Center (YERC), US Fish and Wildlife Service (USFWS) and NASA Terrestrial Observation and Prediction System (TOPS), our study is a partial population viability analysis which aims to establish a link between bat population dynamic and climate before the appearance of WNS. / Biostatistics

Infectious disease dynamics are inherently shaped by the distribution, ecology, and genetic variation of hosts. Conversely, pathogens exert powerful influences on hosts through demographic processes and natural selection. These tenets of disease ecology and evolutionary biology are illustrated in the case of white-nose syndrome (WNS), an emerging infectious disease of hibernating bats. WNS first emerged in 2006 and spread rapidly throughout eastern North America, causing massive declines in bat populations. To understand how host ecology and spatial distribution influence the spread of WNS, I evaluated risk models of colony-level correlates, including bat colony size, species composition, behavior, and gene flow. WNS was more likely to emerge in large colonies first, and species composition and behavior were also significant predictors of risk. Spatial spread was predicted by population genetics of little brown myotis (Myotis lucifugus), indicating coupling of host gene flow and pathogen dispersal, and potential for the application of landscape genetics to predict future spread. To guide management and evaluate pre-existing genetic diversity, I assessed population genetic structure of little brown myotis using restriction site-associated DNA sequencing (RAD-seq). RAD-seq data revealed two populations divided by the Rocky Mountains, with high gene flow between the distributions of putative subspecies. Demographic analyses and genome scans suggest adaptive genetic variation, variation that may be threatened by WNS in eastern North America. Drastic declines from WNS have likely imposed strong selection, and recent stabilization of populations near the disease epicenter suggests that resistance may have evolved in the host population. I generated whole genome sequence data for bats sampled before and after declines to test for demographic changes and natural selection. Average genomic differentiation and nucleotide diversity indicated little demographic change between the two periods, but preliminary analyses suggest genomic regions of differentiation combined with decreased nucleotide diversity in post-WNS relative to pre-WNS samples, hinting at a pattern of natural selection. Additional samples and in-depth analyses are necessary to robustly test these patterns; however, identification of signatures of selection in the bat genome would be an exciting indication of a rapid evolutionary response to an introduced disease.

abstract: Desert ecosystems of the southwest United States are characterized by hot and arid climates, but hibernating bats can be found at high altitudes. The emerging fungal infection, white-nose syndrome, causes mortality in hibernating bat populations across eastern North America and the pathogen is increasingly observed in western regions. However, little is known about the ecology of hibernating bats in the southwest, which can help predict how these populations may respond to the fungus. My study investigated hibernating bats during two winters (2018-2019/2019-2020) at three caves in northern Arizona to: (1) describe diversity and abundance of hibernating bats using visual internal surveys and photographic documentation, (2) determine the duration of hibernation by recording bat echolocation call sequences outside caves and recording bat activity in caves using visual inspection, and (3) describe environmental conditions where hibernating bats are roosting. Adjacent to bats, I collected temperature and relative humidity, which I converted into absolute humidity. I documented hibernation status (i.e. active vs. not active) and roosting body position (i.e. open, partially hidden, and hidden). Between September 2018 and April 2019, 246 bat observations were recorded across the three caves. The majority of bats were identified as Myotis spp. (45.9\%, n=113), followed by Corynorhinus townsendii (45.5\%, n=112), Parastrellus hesperus (4.8\%, n=12), Eptesicus fuscus (3.6\%, n=9). Between September 2019 and April 2020, I documented a total of 361 bat observations across the three caves. C. townsendii was most prevalent (52.9\%, n=191), followed by the category P. hesperus/Myotis spp. (25.7\%, n=93), Myotis spp. (12.4\%, n=45), P. Hesperus (4.4\%, n=16), E. fuscus (3.6\%, n=13) and Unknown (0.8\%, n=3). Average conditions adjacent to bats were, temperature=12.5ºC, relative humidity=53\%, and absolute humidity=4.9 g/kg. Hibernating bats were never observed in large clusters and the maximum hibernating population size was 24, suggesting low risk for pathogen transmission among bats. Hibernation lasted approximately 120 days, with minimal activity documented inside and outside caves. Hibernating bats in northern Arizona may be at low risk for white-nose syndrome based on population size, hibernation length, roosting behavior, and absolute humidity, but other variables (e.g. temperature) indicate the potential for white-nose syndrome impacts on these populations. / Dissertation/Thesis / Masters Thesis Biology 2020

Since white-nose syndrome (WNS) arrived in the northeastern U.S. in 2006, several affected bat species have exhibited marked population declines (> 90%). For areas such as Fort Drum in northern New York that are subject to regulatory mandates because of the presence of the endangered Indiana bat (Myotis sodalis), acoustic monitoring is now likely more effective than traditional capture methodologies. In the summers of 2011 and 2012, I implemented intensive acoustic sampling using Anabat detectors at Fort Drum to develop a summer acoustic monitoring protocol that is both cost efficient and effective at detecting species of high conservation or management interest, such as the Indiana bat and the little brown bat (Myotis lucifugus). Habitat analysis of radio telemetry data and occupancy models of acoustic data were congruent in confirming nocturnal spatial use of forested riparian zones by little brown bats.  Additionally, occupancy models of passive versus active sampling revealed that passive acoustic sampling is preferable to active sampling for detecting declining species in the post-WNS context. Finally, assessment of detection probabilities at various arrays of acoustic detector layouts in an expected area of use revealed that a grid of detectors covering a wide spatial extent was more effective at detecting Indiana and little brown bats than permanent stations, transects, or double transects. My findings suggest that acoustic monitoring can be affectively implemented for monitoring Indiana and little brown bats even in areas of severe decline. Future efforts should be aimed at determining effective sampling designs for additional declining species. / Master of Science