Contact-independent exposure to Rhodococcus rhodochrous DAP96253 volatiles does not improve the survival rate of Myotis lucifugus (little brown bats) affected by White-nose Syndrome.

Since the emergence of White-nose Syndrome, a fungal disease in bats, caused by Pseudogymnoascus destructans, hibernating populations of little brown bats (Myotis lucifugus) have declined by 70-90% within P. destructans positive hibernacula. To reduce the impact of White-nose Syndrome to North American little brown bat populations we evaluated if exposure to volatile organic compounds produced by induced cells from Rhodococcus rhodochrous strain DAP96253 could improve the overwinter survival of bats infected by P. destructans. Two simultaneous field treatment trials were conducted at natural hibernacula located in Rockcastle and Breckinridge counties, Kentucky, USA. A combined total of 120 little brown bats were randomly divided into control groups (n = 60) which were not exposed to volatile organic compounds and treatment groups (n = 60) which were exposed to volatile organic compounds produced by non-growth, fermented cell paste composed of R. rhodochrous strain DAP96253 cells. Cox proportional hazard models revealed a significant decreased survival at the Rockcastle field trial site but not the Breckinridge field site. At the Breckinridge hibernacula, overwinter survival for both treatment and control groups were 60%. At the Rockcastle hibernacula, Kaplan-Meier survival curves indicated significantly increased overwinter survival of bats in the control group (43% survived) compared to the treatment group (20% survived). Although complete inhibition of P. destructans by volatile organic compounds produced by induced R. rhodochrous strain DAP96253 cells was observed in vitro studies, our results suggest that these volatile organic compounds do not inhibit P. destructans in situ and may promote P. destructans growth.

Development of an LC-MS/MS Method for Non-Invasive Biomonitoring of Neonicotinoid and Systemic Herbicide Pesticide Residues in Bat Hair.

With over a quarter of the world's bats species facing extinction, there is a need for ecotoxicological studies to assess if acute and sublethal exposure to newer pesticides such as neonicotinoids and carbonates contribute to population declines. Pesticide exposure studies in bats have been limited to terminal sampling methods, therefore we developed a non-invasive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method utilizing hair trimmings. The hair of big brown bats (Eptesicus fuscus) was collected and pooled by county to assess the best extraction solvent and solid-phase-extraction (SPE) clean-up cartridges. Using the best performing extraction solvent, methanol, and the best performing SPE cartridge, Chromabond HR-X, we developed an optimized multiple reaction monitoring (MRM) LC-MS/MS method for simultaneous determination of 3 neonicotinoids, clothianidin, imidacloprid, and thiamethoxam; 1 carbonate, carbaryl; and 4 systemic herbicides, 2,4-D, atrazine, dicamba, and glyphosate. The optimized protocol yielded the detection of 3-8 of the compounds in the county-level bat hair pools. 2,4-D, glyphosate, and imidacloprid were found in all samples with two of the county-level hair samples having glyphosate concentrations of over 3500 pg/mg of hair. This approach has great potential to facilitate non-terminal ecotoxicological studies assessing the effects of subacute (chronic) pesticide exposure in threatened and endangered bat species and other species experiencing population declines.

COUNTCOLORS, AN R PACKAGE FOR QUANTIFICATION OF THE FLUORESCENCE EMITTED BY PSEUDOGYMNOASCUS DESTRUCTANS LESIONS ON THE WING MEMBRANES OF HIBERNATING BATS.

Pseudogymnoascus destructans colonizes the wing membrane of hibernating bats with the potential to form dense fungal hyphae aggregates within cupping erosions. These fungal cupping erosions emit a characteristic fluorescent orange-yellow color when the wing membrane is transilluminated with 385 nm ultraviolet (UV) light. The purpose of this study was to create and validate the R package, countcolors, for quantifying the distinct orange-yellow UV fluorescence in bat-wing membrane lesions caused by P. destructans. Validation of countcolors was completed by first quantifying the percent area of 20, 2.5 cm2 images. These generated images were of two known pixel colors ranging from 0% to 100% of the pixels. The countcolors package accurately measured the known proportion of a given color in each image. Next, 40, 2.5 cm2 sections of UV transilluminated photographs of little brown bat (Myotis lucifugus) wings were given to a single evaluator. The area of fluorescence was both manually measured and calculated using image analysis software and quantified with countcolors. There was good agreement between the two methods (Pearson's correlation=0.915); however, the manual use of imaging software showed a consistent negative bias. Reproducibility of the analysis methods was tested by providing the same images to naive evaluators who previously never used the software; no significant difference (P=0.099) was found among evaluators. Using the R package countcolors takes less time than does manually measuring the fluorescence in image analysis software, and our results showed that countcolors can improve the accuracy when quantifying the area of P. destructans infection in bat wing-membranes.

Using Deuterium Oxide as a Non-Invasive, Non-Lethal Tool for Assessing Body Composition and Water Consumption in Mammals.

Body condition scoring systems and body condition indices are common techniques used for assessing the health status or fitness of a species. Body condition scoring systems are evaluator dependent and have the potential to be highly subjective. Body condition indices can be confounded by foraging, the effects of body weight, as well as statistical and inferential problems. An alternative to body condition scoring systems and body condition indices is using a stable isotope such as deuterium oxide to determine body composition. The deuterium oxide dilution method is a repeatable, quantitative technique used to estimate body composition in humans, wildlife, and domestic species. Additionally, the deuterium oxide dilution technique can be used to determine the water consumption of an individual animal. Here, we describe the adaption of the deuterium oxide dilution technique for assessing body composition in big brown bats (Eptesicus fuscus) and for assessing water consumption in cats (Felis catis).

White-nose syndrome pathology grading in Nearctic and Palearctic bats.

While white-nose syndrome (WNS) has decimated hibernating bat populations in the Nearctic, species from the Palearctic appear to cope better with the fungal skin infection causing WNS. This has encouraged multiple hypotheses on the mechanisms leading to differential survival of species exposed to the same pathogen. To facilitate intercontinental comparisons, we proposed a novel pathogenesis-based grading scheme consistent with WNS diagnosis histopathology criteria. UV light-guided collection was used to obtain single biopsies from Nearctic and Palearctic bat wing membranes non-lethally. The proposed scheme scores eleven grades associated with WNS on histopathology. Given weights reflective of grade severity, the sum of findings from an individual results in weighted cumulative WNS pathology score. The probability of finding fungal skin colonisation and single, multiple or confluent cupping erosions increased with increase in Pseudogymnoascus destructans load. Increasing fungal load mimicked progression of skin infection from epidermal surface colonisation to deep dermal invasion. Similarly, the number of UV-fluorescent lesions increased with increasing weighted cumulative WNS pathology score, demonstrating congruence between WNS-associated tissue damage and extent of UV fluorescence. In a case report, we demonstrated that UV-fluorescence disappears within two weeks of euthermy. Change in fluorescence was coupled with a reduction in weighted cumulative WNS pathology score, whereby both methods lost diagnostic utility. While weighted cumulative WNS pathology scores were greater in the Nearctic than Palearctic, values for Nearctic bats were within the range of those for Palearctic species. Accumulation of wing damage probably influences mortality in affected bats, as demonstrated by a fatal case of Myotis daubentonii with natural WNS infection and healing in Myotis myotis. The proposed semi-quantitative pathology score provided good agreement between experienced raters, showing it to be a powerful and widely applicable tool for defining WNS severity.

Bat wing biometrics: using collagen-elastin bundles in bat wings as a unique individual identifier.

The ability to recognize individuals within an animal population is fundamental to conservation and management. Identification of individual bats has relied on artificial marking techniques that may negatively affect the survival and alter the behavior of individuals. Biometric systems use biological characteristics to identify individuals. The field of animal biometrics has expanded to include recognition of individuals based upon various morphologies and phenotypic variations including pelage patterns, tail flukes, and whisker arrangement. Biometric systems use 4 biologic measurement criteria: universality, distinctiveness, permanence, and collectability. Additionally, the system should not violate assumptions of capture-recapture methods that include no increased mortality or alterations of behavior. We evaluated whether individual bats could be uniquely identified based upon the collagen-elastin bundles that are visible with gross examination of their wings. We examined little brown bats (Myotis lucifugus), northern long-eared bats (M. septentrionalis), big brown bats (Eptesicus fuscus), and tricolored bats (Perimyotis subflavus) to determine whether the "wing prints" from the bundle network would satisfy the biologic measurement criteria. We evaluated 1,212 photographs from 230 individual bats comparing week 0 photos with those taken at weeks 3 or 6 and were able to confirm identity of individuals over time. Two blinded evaluators were able to successfully match 170 individuals in hand to photographs taken at weeks 0, 3, and 6. This study suggests that bats can be successfully re-identified using photographs taken at previous times. We suggest further evaluation of this methodology for use in a standardized system that can be shared among bat conservationists.

Effects of white-nose syndrome on regional population patterns of 3 hibernating bat species.

Hibernating bats have undergone severe recent declines across the eastern United States, but the cause of these regional-scale declines has not been systematically evaluated. We assessed the influence of white-nose syndrome (an emerging bat disease caused by the fungus Pseudogymnoascus destructans, formerly Geomyces destructans) on large-scale, long-term population patterns in the little brown myotis (Myotis lucifugus), the northern myotis (Myotis septentrionalis), and the tricolored bat (Perimyotis subflavus). We modeled population trajectories for each species on the basis of an extensive data set of winter hibernacula counts of more than 1 million individual bats from a 4-state region over 13 years and with data on locations of hibernacula and first detections of white-nose syndrome at each hibernaculum. We used generalized additive mixed models to determine population change relative to expectations, that is, how population trajectories differed with a colony's infection status, how trajectories differed with distance from the point of introduction of white-nose syndrome, and whether declines were concordant with first local observation of the disease. Population trajectories in all species met at least one of the 3 expectations, but none met all 3. Our results suggest, therefore, that white-nose syndrome has affected regional populations differently than was previously understood and has not been the sole cause of declines. Specifically, our results suggest that in some areas and species, threats other than white-nose syndrome are also contributing to population declines, declines linked to white-nose syndrome have spread across large geographic areas with unexpected speed, and the disease or other threats led to declines in bat populations for years prior to disease detection. Effective conservation will require further research to mitigate impacts of white-nose syndrome, renewed attention to other threats to bats, and improved surveillance efforts to ensure early detection of white-nose syndrome.

Handling and blood collection in the little brown bat (Myotis lucifugus).

Bats are useful animal models for the study of unique physiological mechanisms such as echolocation and sensory integration as well as for research on white-nose syndrome. These studies may involve collecting blood samples from bats. This column describes safe techniques for restraint, weighing, fluid administration and blood collection from the little brown bat (Myotis lucifugus).

Improved Analysis of Long-Term Monitoring Data Demonstrates Marked Regional Declines of Bat Populations in the Eastern United States.

Bats are diverse and ecologically important, but are also subject to a suite of severe threats. Evidence for localized bat mortality from these threats is well-documented in some cases, but long-term changes in regional populations of bats remain poorly understood. Bat hibernation surveys provide an opportunity to improve understanding, but analysis is complicated by bats' cryptic nature, non-conformity of count data to assumptions of traditional statistical methods, and observation heterogeneities such as variation in survey timing. We used generalized additive mixed models (GAMMs) to account for these complicating factors and to evaluate long-term, regional population trajectories of bats. We focused on four hibernating bat species - little brown myotis (Myotis lucifugus), tri-colored bat (Perimyotis subflavus), Indiana myotis (M. sodalis), and northern myotis (M. septentrionalis) - in a four-state region of the eastern United States during 1999-2011. Our results, from counts of nearly 1.2 million bats, suggest that cumulative declines in regional relative abundance by 2011 from peak levels were 71% (with 95% confidence interval of ±11%) in M. lucifugus, 34% (±38%) in P. subflavus, 30% (±26%) in M. sodalis, and 31% (±18%) in M. septentrionalis. The M. lucifugus population fluctuated until 2004 before persistently declining, and the populations of the other three species declined persistently throughout the study period. Population trajectories suggest declines likely resulted from the combined effect of multiple threats, and indicate a need for enhanced conservation efforts. They provide strong support for a change in the IUCN Red List conservation status in M. lucifugus from Least Concern to Endangered within the study area, and are suggestive of a need to change the conservation status of the other species. Our modeling approach provided estimates of uncertainty, accommodated non-linearities, and controlled for observation heterogeneities, and thus has wide applicability for evaluating population trajectories in other wildlife species.