Joseph S. Johnson

The White-Nose Syndrome Transcriptome: Activation of Anti-fungal Host Responses in Wing Tissue of Hibernating Little Brown Myotis.

White-nose syndrome (WNS) in North American bats is caused by an invasive cutaneous infection by the psychrophilic fungus Pseudogymnoascus destructans (Pd). We compared transcriptome-wide changes in gene expression using RNA-Seq on wing skin tissue from hibernating little brown myotis (Myotis lucifugus) with WNS to bats without Pd exposure. We found that WNS caused significant changes in gene expression in hibernating bats including pathways involved in inflammation, wound healing, and metabolism. Local acute inflammatory responses were initiated by fungal invasion. Gene expression was increased for inflammatory cytokines, including interleukins (IL) IL-1β, IL-6, IL-17C, IL-20, IL-23A, IL-24, and G-CSF and chemokines, such as Ccl2 and Ccl20. This pattern of gene expression changes demonstrates that WNS is accompanied by an innate anti-fungal host response similar to that caused by cutaneous Candida albicans infections. However, despite the apparent production of appropriate chemokines, immune cells such as neutrophils and T cells do not appear to be recruited. We observed upregulation of acute inflammatory genes, including prostaglandin G/H synthase 2 (cyclooxygenase-2), that generate eicosanoids and other nociception mediators. We also observed differences in Pd gene expression that suggest host-pathogen interactions that might determine WNS progression. We identified several classes of potential virulence factors that are expressed in Pd during WNS, including secreted proteases that may mediate tissue invasion. These results demonstrate that hibernation does not prevent a local inflammatory response to Pd infection but that recruitment of leukocytes to the site of infection does not occur. The putative virulence factors may provide novel targets for treatment or prevention of WNS. These observations support a dual role for inflammation during WNS; inflammatory responses provide protection but excessive inflammation may contribute to mortality, either by affecting torpor behavior or causing damage upon emergence in the spring.

Social networks of Rafinesque's big-eared bats (Corynorhinus rafinesquii) in bottomland hardwood forests

Abstract Understanding social relationships and organization in colonial bat species can provide valuable insight into species ecology and potentially aid in conservation efforts of rare bat species. We applied social network analysis to describe social relationships and organization in 3 colonies of Rafinesques big-eared bats (Corynorhinus rafinesquii) roosting in bottomland hardwood forests in Kentucky. We radiotracked 48 adult big-eared bats to 64 day-roosts over 549 bat-days during the summers of 2009–2011. We measured homophily, network centralization, density, transitivity, and core–periphery structure of networks of bats sharing common roosts, and we measured degree centrality of nodes (bats or roosts) within networks. Patterns of ties within each colony were homophilous by sex (E-I index = −0.87). Males were consistently the least central nodes in bat networks. Bat network centralization ranged from 1.2% to 40% among colonies, and roost network centralization ranged from 17% to 40%. The colony exhibiting the least centralized and most dense bat network also occupied habitat with low roost availability. This roost network was highly centralized, with bats frequently aggregating at a single roost. The colony with the most centralized and least dense bat network occupied habitat with a greater availability of roosts, resulting in diffuse networks of bats and roosts. Transitivity decreased after young became volant in the colony with highest roost availability. Our findings suggest that social structure in colonies of Rafinesques big-eared bats is affected by the sex of individuals in colonies, reproductive season, and the preponderance of available day-roosting habitat.

Vaccination strategies for managing brucellosis in Yellowstone bison.

Concerns over migratory bison (Bison bison) at Yellowstone National Park transmitting brucellosis (Brucella abortus) to cattle herds on adjacent lands led to proposals for bison vaccination. We developed an individual-based model to evaluate how brucellosis infection might respond under alternate vaccination strategies, including: (1) vaccination of female calves and yearlings captured at the park boundary when bison move outside the primary conservation area; (2) combining boundary vaccination with the remote delivery of vaccine to female calves and yearlings distributed throughout the park; and (3) vaccinating all female bison (including adults) during boundary capture and throughout the park using remote delivery of vaccine. Simulations suggested Alternative 3 would be most effective, with brucellosis seroprevalence decreasing by 66% (from 0.47 to 0.16) over a 30-year period resulting from 29% of the population receiving protection through vaccination. Under this alternative, bison would receive multiple vaccinations that extend the duration of vaccine protection and defend against recurring infection in latently infected animals. The initial decrease in population seroprevalence will likely be slow due to high initial seroprevalence (40-60%), long-lived antibodies, and the culling of some vaccinated bison that were subsequently exposed to field strain Brucella and reacted positively on serologic tests. Vaccination is unlikely to eradicate B. abortus from Yellowstone bison, but could be an effective tool for reducing the level of infection. Our approach and findings have applicability world-wide for managers dealing with intractable wildlife diseases that cross wildlife-livestock and wildlife-human interfaces and affect public health or economic well-being.

Host, Pathogen, and Environmental Characteristics Predict White-Nose Syndrome Mortality in Captive Little Brown Myotis (Myotis lucifugus)

An estimated 5.7 million or more bats died in North America between 2006 and 2012 due to infection with the fungus Pseudogymnoascus destructans (Pd) that causes white-nose syndrome (WNS) during hibernation. The behavioral and physiological changes associated with hibernation leave bats vulnerable to WNS, but the persistence of bats within the contaminated regions of North America suggests that survival might vary predictably among individuals or in relation to environmental conditions. To investigate variables influencing WNS mortality, we conducted a captive study of 147 little brown myotis (Myotis lucifugus) inoculated with 0, 500, 5 000, 50 000, or 500 000 Pd conidia and hibernated for five months at either 4 or 10°C. We found that female bats were significantly more likely to survive hibernation, as were bats hibernated at 4°C, and bats with greater body condition at the start of hibernation. Although all bats inoculated with Pd exhibited shorter torpor bouts compared to controls, a characteristic of WNS, only bats inoculated with 500 conidia had significantly lower survival odds compared to controls. These data show that host and environmental characteristics are significant predictors of WNS mortality, and that exposure to up to 500 conidia is sufficient to cause a fatal infection. These results also illustrate a need to quantify dynamics of Pd exposure in free-ranging bats, as dynamics of WNS produced in captive studies inoculating bats with several hundred thousand conidia may differ from those in the wild.

Foraging Ecology of Long-Legged Myotis (Myotis volans) in North-Central Idaho

Abstract Limited information exists on the foraging ecology of the long-legged myotis (Myotis volans), especially with regard to use of available foraging habitats in large, relatively contiguous forested landscapes. During the summers of 2004 and 2005, we radiotagged adult long-legged myotis (n = 70) in north-central Idaho to estimate the size of home ranges and to evaluate use of available foraging habitats. Size of home range and core areas was measured for individuals with ≥31 locations (n = 30) using the adaptive kernel method, and selection among available foraging habitats was evaluated using Euclidean distance analysis. Home-range estimates did not differ among males, pregnant females, and lactating females (P = 0.52). Core-area estimates also did not differ among males, pregnant females, and lactating females (P = 0.62). Second-order habitat analysis, based on vegetation, showed that home ranges of males (P = 0.01), pregnant females (P = 0.001), and lactating females (P = 0.001) all were closest to stands of medium-diameter trees, that is, trees predominantly 12.7–38.0 cm diameter at breast height (dbh), that also contained larger snags typically used as roosts (X̄ = 54.0 cm dbh; n = 100). Second-order habitat analysis, based on slope position, showed that home ranges of males (P = 0.0001), pregnant females (P = 0.001), and lactating females (P = 0.001) were closest to mid-slope positions. Third-order habitat analysis, based on either vegetation or slope position, did not differ from random use for males, pregnant females, and lactating females. More lepidopterans were captured in black-light traps at mid-slope positions than either upper or lower slope positions. Fecal pellets (n = 171) from 62 long-legged myotis revealed a diet primarily composed of Lepidoptera (49.2% volume, 100% frequency) and Coleoptera (31.1% volume, 100% frequency). Examination of our data demonstrates the importance of forest stand structure, topographic position, and abundance of moths in foraging habitat for long-legged myotis.

Seasonal and Geographic Trends in Acoustic Detection of Tree-Roosting Bats

Migratory routes, timing, and behavior are some of the least studied facets of bat biology, and possibly play roles in bat mortality rates observed at commercial wind energy facilities. We used acoustic detectors to record seasonal activity of silver-haired (Lasionycteris noctivagans), hoary (Lasiurus cinereus), and eastern red (Lasiurus borealis) bats above the forest canopy at one existing and 13 proposed wind energy facilities in seven eastern U.S. states between April and November 2007 and 2008. We correlated species detection rates between surveys located within three predetermined geographic regions, and correlated species detection rates from two survey locations with mortality reported from a nearby commercial wind facility. We identified 2,603 L. noctivagans, 1,908 L. cinereus, and 6,802 L. borealis calls during 6,153 detector-nights. We found a greater number of significant correlations between sites for silver-haired and hoary bat detection rates than in eastern red bat detection rates. Each species exhibited unique seasonal trends in detections among geographic regions. Previously reported mortality rates of L. noctivagans and L. cinereus from a wind energy facility were positively correlated with detection rates of those species at one of our survey locations within 50 km (r = 0.65, P < 0.001 and r = 0.28, P < 0.01, respectively; in both cases d.f. = 94) and with another location within 100 km (r = 0.44, P < 0.001 and r = 0.28, P < 0.01, respectively; in both cases d.f. = 81). These data indicate that seasonal detection rates of all three species under study reflect their different migratory patterns that may be useful in predicting the timing of mortality events at wind energy facilities.

Immune responses in hibernating little brown myotis (Myotis lucifugus) with white-nose syndrome

White-nose syndrome (WNS) is a fungal disease responsible for decimating many bat populations in North America. Pseudogymnoascus destructans (Pd), the psychrophilic fungus responsible for WNS, prospers in the winter habitat of many hibernating bat species. The immune response that Pd elicits in bats is not yet fully understood; antibodies are produced in response to infection by Pd, but they may not be protective and indeed may be harmful. To understand how bats respond to infection during hibernation, we studied the effect of Pd inoculation on the survival and gene expression of captive hibernating Myotis lucifugus with varying pre-hibernation antifungal antibody titres. We investigated gene expression through the transcription of selected cytokine genes (Il6, Il17a, Il1b, Il4 and Ifng) associated with inflammatory, Th1, Th2 and Th17 immune responses in wing tissue and lymph nodes. We found no difference in survival between bats with low and high anti-Pd titres, although anti-Pd antibody production during hibernation differed significantly between infected and uninfected bats. Transcription of Il6 and Il17a was higher in the lymph nodes of infected bats compared with uninfected bats. Increased transcription of these cytokines in the lymph node suggests that a pro-inflammatory immune response to WNS is not restricted to infected tissues and occurs during hibernation. The resulting Th17 response may be protective in euthermic bats, but because it may disrupt torpor, it could be detrimental during hibernation.

Antibodies to Pseudogymnoascus destructans are not sufficient for protection against white‐nose syndrome

White-nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans (Pd) that affects bats during hibernation. Although millions of bats have died from WNS in North America, mass mortality has not been observed among European bats infected by the fungus, leading to the suggestion that bats in Europe are immune. We tested the hypothesis that an antibody-mediated immune response can provide protection against WNS by quantifying antibodies reactive to Pd in blood samples from seven species of free-ranging bats in North America and two free-ranging species in Europe. We also quantified antibodies in blood samples from little brown myotis (Myotis lucifugus) that were part of a captive colony that we injected with live Pd spores mixed with adjuvant, as well as individuals surviving a captive Pd infection trial. Seroprevalence of antibodies against Pd, as well as antibody titers, was greater among little brown myotis than among four other species of cave-hibernating bats in North America, including species with markedly lower WNS mortality rates. Among little brown myotis, the greatest titers occurred in populations occupying regions with longer histories of WNS, where bats lacked secondary symptoms of WNS. We detected antibodies cross-reactive with Pd among little brown myotis naïve to the fungus. We observed high titers among captive little brown myotis injected with Pd. We did not detect antibodies against Pd in Pd-infected European bats during winter, and titers during the active season were lower than among little brown myotis. These results show that antibody-mediated immunity cannot explain survival of European bats infected with Pd and that little brown myotis respond differently to Pd than species with higher WNS survival rates. Although it appears that some species of bats in North America may be developing resistance to WNS, an antibody-mediated immune response does not provide an explanation for these remnant populations.

Effects of Reproductive Condition, Roost Microclimate, and Weather Patterns on Summer Torpor Use by a Vespertilionid Bat

A growing number of mammal species are recognized as heterothermic, capable of maintaining a high-core body temperature or entering a state of metabolic suppression known as torpor. Small mammals can achieve large energetic savings when torpid, but they are also subject to ecological costs. Studying torpor use in an ecological and physiological context can help elucidate relative costs and benefits of torpor to different groups within a population. We measured skin temperatures of 46 adult Rafinesques big-eared bats (Corynorhinus rafinesquii) to evaluate thermoregulatory strategies of a heterothermic small mammal during the reproductive season. We compared daily average and minimum skin temperatures as well as the frequency, duration, and depth of torpor bouts of sex and reproductive classes of bats inhabiting day-roosts with different thermal characteristics. We evaluated roosts with microclimates colder (caves) and warmer (buildings) than ambient air temperatures, as well as roosts with intermediate conditions (trees and rock crevices). Using Akaikes information criterion (AIC), we found that different statistical models best predicted various characteristics of torpor bouts. While the type of day-roost best predicted the average number of torpor bouts that bats used each day, current weather variables best predicted daily average and minimum skin temperatures of bats, and reproductive condition best predicted average torpor bout depth and the average amount of time spent torpid each day by bats. Finding that different models best explain varying aspects of heterothermy illustrates the importance of torpor to both reproductive and nonreproductive small mammals and emphasizes the multifaceted nature of heterothermy and the need to collect data on numerous heterothermic response variables within an ecophysiological context.

Geographic Variation in Roost-Site Selection of Long-Legged Myotis in the Pacific Northwest

Abstract Several species of bats in the Pacific Northwest of the United States, including long-legged myotis (Myotis volans), are dependent on snags in coniferous forests during summer for roosting and rearing young. Thus, data on roosting preferences of this species are needed to integrate their habitat requirements into shifting plans for management of forests in this region. Therefore, from 2001 to 2006, we radiotracked adult female long-legged myotis (n  =  153) to day roosts (n  =  395) across 6 watersheds in Washington, Oregon, and Idaho, USA, and compared characteristics associated with roosting sites to those of random snags (n  =  260) sampled in the same watersheds using use–availability logistic regression and an information-theoretic approach. Model rankings varied among geographic locations, with quantity of stem surface for roosting the best model for explaining roost-site selection of long-legged myotis in both Washington and Oregon. Model rankings for populations of bats in Idaho found stand- and landscape-scale features to be important in roost-site selection, with a habitat fragmentation model and a foraging habitat quality model both demonstrating strong support as best model. Choice of day roosts by long-legged myotis was associated with snags that were taller, intact at the top of the stem, possessing a greater amount of exfoliating bark, in stands with a larger basal area of dead stems, and in landscapes that were unfragmented (i.e., supporting lesser amounts of edge). Results indicate that roost-site selection of bats in western coniferous forests, particularly long-legged myotis, is likely to be region-specific. We encourage land managers to consider importance of geographic variation in intraspecific habitat use in forest-dwelling bats when implementing silvicultural systems to promote biological diversity in actively managed forests of the Pacific Northwest region.