Disturbance of hibernating bats due to researchers entering caves to conduct hibernacula surveys.

Estimating population changes of bats is important for their conservation. Population estimates of hibernating bats are often calculated by researchers entering hibernacula to count bats; however, the disturbance caused by these surveys can cause bats to arouse unnaturally, fly, and lose body mass. We conducted 17 hibernacula surveys in 9 caves from 2013 to 2018 and used acoustic detectors to document cave-exiting bats the night following our surveys. We predicted that cave-exiting flights (i.e., bats flying out and then back into caves) of Townsend's big-eared bats (Corynorhinus townsendii) and western small-footed myotis (Myotis ciliolabrum) would be higher the night following hibernacula surveys than on nights following no surveys. Those two species, however, did not fly out of caves more than predicted the night following 82% of surveys. Nonetheless, the activity of bats flying out of caves following surveys was related to a disturbance factor (i.e., number of researchers × total time in a cave). We produced a parsimonious model for predicting the probability of Townsend's big-eared bats flying out of caves as a function of disturbance factor and ambient temperature. That model can be used to help biologists plan for the number of researchers, and the length of time those individuals are in a cave to minimize disturbing bats.

Year-round Observations of Adult Culex Mosquitoes in Storm Sewer Pipes in the Northwestern Chicago Suburbs.

Over the course of three years, 200 ft to 0.75 mi (60 m to 1.2 km) sections of 3 larger (>6 ft [1.8 m] diam) belowground storm sewer conveyance pipes in the northwestern Chicago suburbs were inspected for the presence of adult mosquitoes. Culex mosquitoes were by far the most common (555 of 556 [99.8%] total mosquitoes) collected within pipes during all four meteorological seasons (i.e. during months of October, January, May, August). These observations support prior work elsewhere, suggesting storm sewer pipes are consistent sites of refuge for adult Culex mosquitoes.

Year-round Observations of Adult Culex Mosquitoes in Storm Sewer Pipes in the Northwestern Chicago Suburbs.

Over the course of three years, 200 ft to 0.75 mi (60 m to 1.2 km) sections of 3 larger (>6 ft [1.8 m] diam) belowground storm sewer conveyance pipes in the northwestern Chicago suburbs were inspected for the presence of adult mosquitoes. Culex mosquitoes were by far the most common (555 of 556 [99.8%] total mosquitoes) collected within pipes during all four meteorological seasons (i.e. during months of October, January, May, August). These observations support prior work elsewhere, suggesting storm sewer pipes are consistent sites of refuge for adult Culex mosquitoes.

Phenology and predictors of spring emergence for the Timber Rattlesnake (Crotalus horridus).

Many temperate reptiles survive winter by using subterranean refugia until external conditions become suitable for activity. Determining when to emerge from refugia relies on the ability to interpret when above-ground environmental conditions are survivable. If temperate reptiles rely on specific environmental cues such as temperature to initiate emergence, we should expect emergence phenologies to be predictable using local climatic data. However, specific predictors of emergence for many temperate reptiles, including the Timber Rattlesnake (Crotalus horridus), remain unclear, limiting our understanding of their overwintering phenology and restricting effective conservation and management. Our objectives were to identify environmental cues of spring emergence for C. horridus in Illinois to determine the species' emergence phenology, and to examine the applicability of identified cues in predicting emergence phenology across the species' range. We used wildlife cameras and weather station-derived environmental data to observe and predict the daily surface presence of C. horridus throughout the late winter and early spring at communal refugia in west-central and northern Illinois. The most parsimonious model for predicting surface presence included the additive effects of maximum daily temperature, accumulated degree days, and latitude. With a notable exception in the southeastern U.S., the model accurately predicted the average emergence day for eight other populations range wide, emphasizing the importance of temperature in influencing the phenological plasticity observed across the species' range. The apparent broad applicability of the model to other populations suggests it can be a valuable tool in predicting spring emergence phenology. Our results provide a foundation for further ecological enquiries and improved management and conservation strategies.

Assessing the threat of bat-associated fungal pathogens.

Fungal pathogens have become an increasingly important topic in recent decades. Yet whilst various cankers and blights have gained attention in temperate woodlands and crops, the scope for fungal pathogens of animals and their potential threat has received far less attention. With a shifting climate, the threat from fungal pathogens is predicted to increase in the future, thus understanding the spread of fungi over landscapes as well as taxa that may be at risk is of particular importance. Cave ecosystems provide potential refugia for various fungi, and roosts for bats. With their well vascularized wings and wide-ranging distributions, bats present potential fungal vectors. Furthermore, whilst bat immune systems are generally robust to bacterial and viral pathogens, they can be susceptible to fungal pathogens, particularly during periods of stress such as hibernation. Here we explore why bats are important and interesting vectors for fungi across landscapes and discuss knowledge gaps that require further research.

COULD WHITE-NOSE SYNDROME MANIFEST DIFFERENTLY IN MYOTIS LUCIFUGUS IN WESTERN VERSUS EASTERN REGIONS OF NORTH AMERICA? A REVIEW OF FACTORS.

White-nose syndrome (WNS) has notably affected the abundance of Myotis lucifugus (little brown myotis) in North America. Thus far, substantial mortality has been restricted to the eastern part of the continent where the cause of WNS, the invasive fungus Pseudogymnoascus destructans, has infected bats since 2006. To date, the state of Washington is the only area in the Western US or Canada (the Rocky Mountains and further west in North America) with confirmed cases of WNS in bats, and there the disease has spread more slowly than it did in Eastern North America. Here, we review differences between M. lucifugus in western and eastern parts of the continent that may affect transmission, spread, and severity of WNS in the West and highlight important gaps in knowledge. We explore the hypothesis that western M. lucifugus may respond differently to WNS on the basis of different hibernation strategies, habitat use, and greater genetic structure. To document the effect of WNS on M. lucifugus in the West most effectively, we recommend focusing on maternity roosts for strategic disease surveillance and monitoring abundance. We further recommend continuing the challenging work of identifying hibernation and swarming sites to better understand the microclimates, microbial communities, and role in disease transmission of these sites, as well as the ecology and hibernation physiology of bats in noncavernous hibernacula.

Cooling of bat hibernacula to mitigate white-nose syndrome.

White-nose syndrome (WNS) is a fungal disease that has caused precipitous declines in several North American bat species, creating an urgent need for conservation. We examined how microclimates and other characteristics of hibernacula have affected bat populations following WNS-associated declines and evaluated whether cooling of warm, little-used hibernacula could benefit bats. During the period following mass mortality (2013-2020), we conducted 191 winter surveys of 25 unmanipulated hibernacula and 6 manipulated hibernacula across Pennsylvania (USA). We joined these data with additional datasets on historical (pre-WNS) bat counts and on the spatial distribution of underground sites. We used generalized linear mixed models and model selection to identify factors affecting bat populations. Winter counts of Myotis lucifugus were higher and increased over time in colder hibernacula (those with midwinter temperatures of 3-6 °C) compared with warmer (7-11 °C) hibernacula. Counts of Eptesicus fuscus, Myotis leibii, and Myotis septentrionalis were likewise higher in colder hibernacula (temperature effects = -0.73 [SE 0.15], -0.51 [0.18], and -0.97 [0.28], respectively). Populations of M. lucifugus and M. septentrionalis increased most over time in hibernacula surrounded by more nearby sites, whereas Eptesicus fuscus counts remained high where they had been high before WNS onset (pre-WNS high count effect = 0.59 [0.22]). Winter counts of M. leibii were higher in hibernacula with high vapor pressure deficits (VPDs) (particularly over 0.1 kPa) compared with sites with lower VPDs (VPD effect = 15.3 [4.6]). Counts of M. lucifugus and E. fuscus also appeared higher where VPD was higher. In contrast, Perimyotis subflavus counts increased over time in relatively warm hibernacula and were unaffected by VPD. Where we manipulated hibernacula, we achieved cooling of on average 2.1 °C. At manipulated hibernacula, counts of M. lucifugus and P. subflavus increased over time (years since manipulation effect = 0.70 [0.28] and 0.51 [0.15], respectively). Further, there were more E. fuscus where cooling was greatest (temperature difference effect = -0.46 [SE 0.11]), and there was some evidence there were more P. subflavus in hibernacula sections that remained warm after manipulation. These data show bats are responding effectively to WNS through habitat selection. In M. lucifugus, M. septentrionalis, and possibly P. subflavus, this response is ongoing, with bats increasingly aggregating at suitable hibernacula, whereas E. fuscus remain in previously favored sites. Our results suggest that cooling warm sites receiving little use by bats is a viable strategy for combating WNS.

El síndrome de nariz blanca (SNB) es una enfermedad fúngica que ha causado declinaciones precipitadas en varias especies de murciélagos norteamericanos, creando una necesidad urgente por conservarlas. Analizamos cómo los microclimas y otras características de los hibernáculos han afectado a las poblaciones de murciélagos después de declinaciones asociadas al SNB y evaluamos si el enfriamiento de hibernáculos cálidos con poco uso podría beneficiar a los murciélagos. Durante el periodo posterior a una mortalidad masiva (2013 - 2020), realizamos 191 censos invernales en 25 hibernáculos sin manipulación y en seis hibernáculos manipulados localizados en Pensilvania (EUA). Juntamos estos datos con conjuntos adicionales de datos de los conteos históricos (previos WNS) de murciélagos y de la distribución espacial de sitios subterráneos. Usamos modelos mixtos lineales generalizados y selección de modelos para identificar los factores que afectan a las poblaciones de murciélagos. Los conteos invernales de Myotis lucifugus fueron más altos e incrementaron con el tiempo en los hibernáculos fríos (aquellos con temperaturas de 3 - 6° C registradas a mitad del invierno) en comparación con los hibernáculos cálidos (7 - 11° C). Los conteos Eptesicus fuscus, M. leibii, y M. septentrionalis fueron igualmente más altos en los hibernáculos fríos (efectos de la temperatura = -0.73 [ES 0.15], -0.51 [0.18], y -0.97 [0.28], respectivamente). Las poblaciones de M. lucifugus y M. septentrionalis fueron las que más incrementaron con el tiempo en los hibernáculos rodeados por más sitios cercanos, mientras que los conteos de E. fuscus permanecieron altos en donde ya habían sido altos antes del comienzo del SNB (el efecto del conteo alto previo al SNB = 0.59 [0.22]). Los conteos invernales de M. leibii fueron más altos en los hibernáculos con altos déficits de presión de vapor (DPV) (particularmente por encima de los 0.1 kPa) en comparación con los sitios con un DPV menor (efecto del VPD = 15.3 [4.6]). Los conteos de M. lucifugus y E. fuscus también fueron más altos en donde el DPV era alto. Al contrario, los conteos de Perimyotis subflavus incrementaron con el tiempo en hibernáculos relativamente cálidos y no se vieron afectados por el DPV. En donde alcanzamos un promedio de enfriamiento de 2.1° C de los hibernáculos, los conteos de M. lucifugus y P. subflavus incrementaron con el tiempo (años desde el efecto de manipulación = 0.70 [0.28] y 0.51 [0.15], respectivamente). Además, encontramos más E. fuscus en donde el enfriamiento fue mayor (efecto de la diferencia en temperatura = −0.46 [ES 0.11]), y hubo algunas evidencias de que había mayor cantidad de P. subflavus en las secciones del hibernáculo que permanecieron cálidas después de la manipulación. Estos datos muestran que los murciélagos están respondiendo efectivamente al SNB mediante la selección de hábitat. En el caso de M. lucifugus, M. septentrionalis y posiblemente P. subflavus, esta respuesta es persistente, con los murciélagos agrupándose cada vez más en hibernáculos adecuados, mientras que E. fuscus permanece en sitios favorecidos previamente. Nuestros resultados sugieren que el enfriamiento de los sitios cálidos que reciben poco uso por parte de los murciélagos es una estrategia viable para combatir al SNB. Enfriamiento de los Hibernáculos de Murciélagos para Mitigar el Síndrome de Nariz Blanca.

The Milieu Souterrain Superficiel as hibernation habitat for bats: implications for white-nose syndrome.

Recent studies have revealed that western populations of little brown bats (Myotis lucifugus) in North America exhibit different hibernation behavior than their eastern counterparts. Understanding these differences is essential for assessing the risk white-nose syndrome (WNS) poses to western bat populations. We used acoustic monitoring and radiotelemetry to study the overwintering behavior of little brown bats near Juneau, Alaska during 2011-2014. Our objectives were to identify the structures they use for hibernation, measure the microclimates within those structures, and determine the timing of immergence and emergence and the length of the hibernation season. We radiotracked 10 little brown bats to underground hibernacula dispersed along two ridge systems. All hibernacula were ≤ 24.2 km from where the bats were captured. Eight bats hibernated in the "Milieu Souterrain Superficiel" (MSS), a network of air-filled underground voids between the rock fragments found in scree (talus) deposits. Two bats hibernated in holes in the soil beneath the root system of a tree or stump (rootball). At least two hibernacula in the MSS were reused in subsequent years. Average MSS and rootball temperatures were warmer and more stable than ambient temperature and were well below the optimal growth range of the fungus that causes WNS. Temperatures in the MSS dropped below freezing, but MSS temperatures increased with depth, indicating bats could avoid subfreezing temperatures by moving deeper into the MSS. Relative humidity (RH) approached 100% in the MSS and under rootballs and was more stable than ambient RH, which also was high, but dropped substantially during periods of extreme cold. Acoustic monitoring revealed that bats hibernated by late October and began emerging by the second week of April; estimates of minimum length of the hibernation season ranged from 156 to 190 days. The cold temperatures, dispersed nature of the hibernacula, and close proximity of hibernacula to summering areas may slow the spread and reduce the impacts of WNS on local populations of little brown bats.

White-Nose Syndrome Confirmed in Italy: A Preliminary Assessment of Its Occurrence in Bat Species.

Although no mass mortality has been recorded so far, the precise demographic effect of white-nose syndrome (WNS) on European bats still remains to be ascertained. Following the first isolation of P. destructans in Italy, further surveys were performed to assess the distribution of the fungus in NW Italy and its effects on bats. Data were collected from March 2019 to April 2020 at sites used for hibernation (six sites) and/or for reproduction (four sites) in Piedmont and Aosta Valley. A total of 138 bats, belonging to 10 species, were examined to identify clinical features possibly related to the fungal presence. Culture from swabs and the molecular identification of isolates confirmed the presence of P. destructans in bats from five sites, including two maternal roosts. Dermal fungal infiltration, the criterion to assess the presence of WNS, was observed in biopsies of bats belonging to Myotis blythii, M. daubentonii, M. emarginatus and M. myotis. This is the first report of the disease in Italy. The results suggest a greater susceptibility to the infection of the genus Myotis and particularly of M. emarginatus, possibly due to the long length of its hibernation period. Other fungal dermatophytes were also observed.

Generational shift in the migratory common noctule bat: first-year males lead the way to hibernacula at higher latitudes.

Many migratory species have shifted their geographic distribution in response to climate change, yet the underlying mechanisms are poorly understood, particularly for mammals. We hypothesized that generational shifts are underlying the observed colonization of hibernation sites further north in a migratory bat, the common noctule (Nyctalus noctula). To evaluate our hypothesis, we collected long-term data on the migratory status and demography of common noctules in a recently colonized hibernation area. Based on isotopic data of 413 individuals, we observed a significant decline in the proportion of long-distance migrants from 2004 to 2015 for both sexes and across all age groups. Demographic data collected between 2007 and 2016 from 3394 individuals demonstrated that subadult males were more abundant during the early colonization stage, followed by a gradual shift to a more balanced age and sex composition. Our results suggest that the colonization of hibernacula at higher latitudes is promoted by generational shifts, involving mostly first-year males. Generational shifts seem to be a likely mechanism for distribution changes in other bats and potentially also in other mammals.

Over-Winter Survival and Nest Site Selection of the West-European Hedgehog (Erinaceus europaeus) in Arable Dominated Landscapes.

The West-European hedgehog (Erinaceus europaeus) has declined markedly in the UK. The winter hibernation period may make hedgehogs vulnerable to anthropogenic habitat and climate changes. Therefore, we studied two contrasting populations in England to examine patterns of winter nest use, body mass changes and survival during hibernation. No between-site differences were evident in body mass prior to hibernation nor the number of winter nests used, but significant differences in overwinter mass change and survival were observed. Mass change did not, however, affect survival rates; all deaths occurred prior to or after the hibernation period, mainly from predation or vehicle collisions. Hedgehogs consistently nested in proximity to hedgerows, roads and woodlands, but avoided pasture fields; differences between sites were evident for the selection for or avoidance of arable fields, amenity grassland and buildings. Collectively, these data indicate that hibernation was not a period of significant mortality for individuals that had attained sufficient weight (>600 g) pre-hibernation. Conversely, habitat composition did significantly affect the positioning of winter nests, such that different land management practices (historic and current) might potentially influence hibernation success. The limitations of this study and suggestions for future research are discussed.

Genotyping-in-Thousands by sequencing reveals marked population structure in Western Rattlesnakes to inform conservation status.

Delineation of units below the species level is critical for prioritizing conservation actions for species at-risk. Genetic studies play an important role in characterizing patterns of population connectivity and diversity to inform the designation of conservation units, especially for populations that are geographically isolated. The northernmost range margin of Western Rattlesnakes (Crotalus oreganus) occurs in British Columbia, Canada, where it is federally classified as threatened and restricted to five geographic regions. In these areas, Western Rattlesnakes hibernate (den) communally, raising questions about connectivity within and between den complexes. At present, Western Rattlesnake conservation efforts are hindered by a complete lack of information on genetic structure and degree of isolation at multiple scales, from the den to the regional level. To fill this knowledge gap, we used Genotyping-in-Thousands by sequencing (GT-seq) to genotype an optimized panel of 362 single nucleotide polymorphisms (SNPs) from individual samples (n = 461) collected across the snake's distribution in western Canada and neighboring Washington (USA). Hierarchical STRUCTURE analyses found evidence for population structure within and among the five geographic regions in BC, as well as in Washington. Within these regions, 11 genetically distinct complexes of dens were identified, with some regions having multiple complexes. No significant pattern of isolation-by-distance and generally low levels of migration were detected among den complexes across regions. Additionally, snakes within dens generally were more related than those among den complexes within a region, indicating limited movement. Overall, our results suggest that the single, recognized designatable unit for Western Rattlesnakes in Canada should be re-assessed to proactively focus conservation efforts on preserving total genetic variation detected range-wide. More broadly, our study demonstrates a novel application of GT-seq for investigating patterns of diversity in wild populations at multiple scales to better inform conservation management.

Trichoderma polysporum selectively inhibits white-nose syndrome fungal pathogen Pseudogymnoascus destructans amidst soil microbes.

BACKGROUND: Pseudogymnoascus destructans (Pd), the causative fungal agent of white-nose syndrome (WNS), has led to the deaths of millions of hibernating bats in the United States of America (USA) and Canada. Efficient strategies are needed to decontaminate Pd from the bat hibernacula to interrupt the disease transmission cycle without affecting the native microbes. Previously, we discovered a novel Trichoderma polysporum (Tp) strain (WPM 39143), which inhibited the growth of Pd in autoclaved soil samples. In the present investigation, we used culture-based approaches to determine Tp-induced killing of native and enriched Pd in the natural soil of two bat hibernacula. We also assessed the impact of Tp treatment on native microbial communities by metagenomics. RESULTS: Our results demonstrated that Tp at the concentration of 105 conidia/g soil caused 100% killing of native Pd in culture within 5 weeks of incubation. A 10-fold higher concentration of Tp (106 conidia/g soil) killed an enriched Pd population (105 conidia/g soil). The 12,507 fungal operational taxonomic units (OTUs, dominated by Ascomycota and Basidiomycota) and 27,427 bacterial OTUs (dominated by Acidobacteria and Proteobacteria) comprised the native soil microbes of the two bat hibernacula. No significant differences in fungal and bacterial relative abundances were observed between untreated and Tp-treated soil (105 Tp conidia/g soil, p ≤ 0.05). CONCLUSIONS: Our results suggest that Tp-induced killing of Pd is highly specific, with minimal to no impact on the indigenous microbes present in the soil samples. These findings provide the scientific rationale for the field trials of Tp in the WNS-affected hibernacula for the effective decontamination of Pd and the control of WNS.

Determinants of Pseudogymnoascus destructans within bat hibernacula: implications for surveillance and management of white-nose syndrome.

1. Fungal diseases are an emerging global problem affecting human health, food security and biodiversity. Ability of many fungal pathogens to persist within environmental reservoirs can increase extinction risks for host species and presents challenges for disease control. Understanding factors that regulate pathogen spread and persistence in these reservoirs is critical for effective disease management. 2. White-nose syndrome (WNS) is a disease of hibernating bats caused by Pseudogymnoascus destructans (Pd), a fungus that establishes persistent environmental reservoirs within bat hibernacula, which contribute to seasonal disease transmission dynamics in bats. However, host and environmental factors influencing distribution of Pd within these reservoirs are unknown. 3. We used model selection on longitudinally collected field data to test multiple hypotheses describing presence-absence and abundance of Pd in environmental substrates and on bats within hibernacula at different stages of WNS. 4. First detection of Pd in the environment lagged up to one year after first detection on bats within that hibernaculum. Once detected, the probability of detecting Pd within environmental samples from a hibernaculum increased over time and was higher in sediment compared to wall surfaces. Temperature had marginal effects on the distribution of Pd. For bats, prevalence and abundance of Pd were highest on Myotis lucifugus and on bats with visible signs of WNS. 5. Synthesis and applications. Our results indicate that distribution of Pseudogymnoascus destructans (Pd) within a hibernaculum is driven primarily by bats with delayed establishment of environmental reservoirs. Thus, collection of samples from Myotis lucifugus, or from sediment if bats cannot be sampled, should be prioritized to improve detection probabilities for Pd surveillance. Long-term persistence of Pd in sediment suggests that disease management for white-nose syndrome should address risks of sustained transmission from environmental reservoirs.

Density regulation in toad populations (Epidalea calamita, Bufotes viridis) by differential winter survival of juveniles.

The size of amphibian populations varies considerably between years, so that systematic trends in dynamics are difficult to detect. Informed conservation management of presumably declining populations requires the identification of the most sensitive life stage. In temperate-zone anurans there is growing evidence that juveniles hibernating for the first time suffer from substantial winter losses. In two syntopic toads (Epidalea calamita, Bufotes viridis) we monitored survival of such juveniles during four consecutive winters in the natural habitat and in four temperature treatments (3°, 5 °C, 10°/15 °C or 20 °C, natural light-dark cycle) in temperature-controlled chambers during winter. Specifically, we tested the hypotheses that (1) winter mortality of juvenile toads which hibernate for the first time in their life is an important component of population dynamics, and that (2) mortality rates differed between the two species. Parameters quantified were size-dependent winter mortality and body condition of pre- and post-hibernating juveniles. Field data provided evidence for the important role of winter mortality of first-hibernators in population dynamics. Choice of hibernacula differed in E. calamita between small and medium-sized individuals and also between the two species suggesting distinct mortality risks. The inability of small E. calamita to reach frost-proof hibernacula by burrowing, and the exposure of small B. viridis to predators are the most probable causes of size-assortative winter mortality. In conclusion, E. calamita juveniles may benefit from rising average winter temperatures in the future by decreased risk of freezing to death, whereas predator-caused winter mortality of B. viridis juveniles will also depend on the effects of climate warming on predator phenology.

HIERARCHICAL PATTERNS OF GENETIC POPULATION STRUCTURE IN BLACK RAT SNAKES (ELAPHE OBSOLETA OBSOLETA) AS REVEALED BY MICROSATELLITE DNA ANALYSIS.

We investigated the distribution of variation at six microsatellite loci in the black rat snake (Elaphe obsoleta obsoleta). Sampling occurred at three hierarchical scales ranging from communal hibernacula to regional populations, with most locales situated within the Frontenac Axis region of eastern Ontario. We detected no statistically significant pairwise differentiation (FST and RST ) between hibernacula within the same subpopulations (interhibernaculum distance <6 km). However, isolation-by-distance was evident among locales within the Frontenac Axis (maximum of 50 km) and among regional populations (maximum of 1500 km). Conservative estimates of Nc derived from heterozygosity values ranged from approximately 600 to 2000. These values suggest relatively large genetic neighborhoods encompassing many communal hibernacula. Our results considered together suggest viscosity of gene flow over relatively short distances (tens of kilometers), but substantial genetic exchange among local hibernacula.