Life history of many animals is often concentrated around central or focal places such as roosts or breeding sites that impose spatial constraints and shape habitat use. Central place behaviors vary depending on life stages, environmental conditions, and the annual cycle of a species—highlighting the need to consider these variations in management plans. The objective of our study was to contrast central place behaviors of little brown bats (Myotis lucifugus) during summer around a maternity roost, while lactating females raised their pups, and during pre-hibernation swarming (mating) at a cave for both males and females. Given the differences in energetic constraints between seasons and sexes, we hypothesized that return rates, activity, distribution, and habitat use would differ between the maternity roost and the hibernaculum and that male and female behavior would differ. We used an automated telemetry network consisting of 10 receiver towers at each site to track bat movements in the surrounding area. This system recorded over 370,000 detections from 23 lactating females at the maternity roost and over 90,000 detections from 23 males and 15 females at the hibernaculum. The maternity roost acted as a typical central place with females returning to the roost on 81 ± 29% of nights and with activity concentrated in a ∼5 km radius of the roost, primarily along a riparian corridor. At the swarming site, males and females returned on only 22 ± 27% of nights, although when they returned males stayed longer than females. Bats were detected up to 13 km from the swarming site and males were overall detected more frequently than females, suggesting that the spatial extent of use differs between sexes. Our study is one of the first to automatically track bats around both a maternity roost and a hibernaculum, and we offer practical suggestions to improve study designs—particularly to address seasonal shifts and sex-specific behavioral variation. We also highlight the importance of integrating seasonal perspectives to better support ­management plans for bats.

Much of the research into white-nose disease has focused on the hibernation period, while the pathogenic fungus Pseudogymnoascus destructans is actively infecting the bat host. Previous research has found large differences between the susceptible North American Myotis lucifugus and the tolerant European Myotis myotis, suggestive of immunopathology in the former, and a beneficial lack of strong response in the latter. Here, we examined gene expression in these species both during the late-hibernation period and a month after emergence from hibernation, during healing from infection. We utilised paired sampling, collecting wing tissue that was positive and negative for fungal infection fluorescence, to examine changes in whole-transcriptome gene expression that were local to sites of infection at two time points: pre-emergence and 30 days post-emergence from hibernation. Positive samples were contrasted between the two time points to examine longitudinal changes. During the pre-emergence period, local inflammatory responses were observed in both M. myotis and M. lucifugus. Immune responses between the tolerant and susceptible species were dissimilar, favouring Th1 and Th17 cytokine responses, respectively. This lends weight to immunopathology as a contributing factor to mortality in M. lucifugus. Continual immune responses may not only contribute to immunopathology and host mortality but also have important carry-over effects on reproduction and subsequent pre-winter fattening, affecting population viability over a longer period of time than previously considered.

ABSTRACTParasites are an abundant and diverse group of organisms that are often excluded from biodiversity surveys, limiting our understanding of host–parasite relationships and parasite diversity. Parasites are dependent on their hosts for survival and parasite populations are at risk if their host populations decline. The aim of our study was to quantify and compare the ectoparasite communities of two sympatric Atlantic Canadian bat species, Myotis lucifugus and M. septentrionalis. Ectoparasites were collected from bats captured for research throughout Atlantic Canada between 1999 and 2017 during the active season (May–October). The prevalence and mean intensity of infection were calculated for each identified ectoparasite species and generalized linear mixed models were used to assess host differences in infection by the two most abundant ectoparasites. Both bat species hosted ectoparasite communities dominated by the mite Spinturnix americanus and the flea Myodopsylla insignis with other ectoparasites being rarely encountered. Despite being the most common ectoparasites of both bat species, our results suggest that infections of these ectoparasites vary between them with M. insignis prevalence being greater on M. lucifugus and S. americanus prevalence being greater on M. septentrionalis. We suggest these differences in infection burden are related to the social and roosting behaviors of these hosts and the life history of the ectoparasites. Monitoring parasites concurrently with focal species is important for capturing this aspect of biodiversity and for understanding how host–parasite dynamics may be disrupted if hosts undergo drastic demographic changes.

ABSTRACTDisease outcomes result from the interaction between host, pathogen, and environmental factors. Understanding how these components interact to influence spatial and temporal variations in disease severity can enhance our insights into the drivers of disease outbreaks, ultimately improving our ability to mitigate the impact of disease through better forecasts and management actions. White‐nose syndrome (WNS) in bats, caused by the fungal pathogen Pseudogymnoascus destructans (Pd), has been detected in hibernating bats across much of the United States and Canada. This pathogen has led to widespread population declines in some bat species, for example, Myotis lucifugus; however, not all infected populations exhibit similar decreases in numbers. Despite long‐term detection and high infection levels, the population of M. lucifugus that uses Tippy Dam, in northern Michigan, as a hibernaculum has not experienced a decline compared to other populations in the state. To assess local population effects that may contribute to reduced disease severity at Tippy Dam, we brought 30 hibernating M. lucifugus from Tippy Dam and 30 from a geographically similar hibernaculum with a history of declines from WNS into captivity at the U.S. Geological Survey, National Wildlife Health Center. We challenged the bats with a Pd inoculum and monitored survival, pathology, and Pd loads for up to 120 days. This allowed us to remove local environmental effects that could influence WNS disease severity. We observed no effect of source population on either survival or wing damage from Pd infection. Our results suggest that population persistence and lowered disease severity in Tippy Dam are likely driven by local environmental factors found within the dam. As Pd continues to spread westward, understanding environmental factors that influence the severity of Pd infection in hibernating bats has the potential to guide management decisions and help predict the survival of susceptible bat species in the western United States.

Modeling and analysis of maize agroecosystem dynamics with stresses

Transgenic animals are invaluable tools in genetic studies, disease modeling, drug discovery, and biotechnology. However, the low efficiency of transgenic animal generation can be an obstacle to their application. Here, we report the generation of transgenic mice using PBatase, the piggyBat transposase from the little brown bat (Myotis lucifugus). PBatase exhibits detectable transposition activity in fertilized mouse eggs within a limited concentration range, although the overall activity was lower than that of PBase, the piggyBac transposase from the cabbage looper moth (Trichoplusia ni). Transgenic animals carrying low transgene copy numbers were successfully generated with high efficiency using PBatase, and the transgene was subsequently transmitted to the next generation. This technique will be useful for the generation of transgenic animals carrying single copies of a transgene.

Since 2007, white-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans, has killed millions of bats across North America by disrupting hibernation cycles, causing premature fat depletion and starvation. Little brown bats (Myotis lucifugus) from some populations persisting after WNS store larger pre-hibernation fat reserves than bats did before WNS, which may help bats survive winter starvation and mount an immune response to Pd in spring. MicroRNAs (miRNAs) are highly conserved, small, non-coding RNA molecules that regulate gene expression post-transcriptionally. Aberrant miRNA expression can affect metabolic pathways in mammals and has been linked to various diseases. If fat reserves and immune mechanisms influence survival from WNS, then miRNAs regulating metabolic and immune-related genes might affect WNS pathogenesis and bat survival. A previous study identified 43 miRNAs differentially expressed in bats with WNS. We analyzed these miRNAs for their roles in metabolism and immune-related pathways, using DIANA Tools and KEGG analysis, to determine a subset that could serve as biomarkers of pathophysiology or survival in WNS-affected bats. We identified miR-543, miR-27a, miR-92b, and miR-328 as particularly important because they regulate multiple pathways likely important for WNS (i.e., immune response, lipogenesis, insulin signaling, and FOXO signaling). As proof-of-concept, we used reverse transcription quantitative real-time PCR (RT-qPCR) to quantify the prevalence of these miRNAs in plasma samples of bats (n = 11) collected from a post-WNS population during fall fattening. All the selected miRNAs were detectable in at least some bats during fall fattening although prevalence varied among miRNAs. Future in vivo validation studies would help confirm functional roles and biomarker utility of these miRNAs for WNS-affected bats.

Bats are a taxa of high conservation concern and are facing numerous threats including widespread mortality due to White-Nose Syndrome (WNS) in North America. With this decline comes increasing difficulty in monitoring imperiled bat species due to lower detection probabilities of both mist-netting and acoustic surveys. Lure technology shows promise to increase detection while decreasing sampling effort; however, to date research has primarily focused on increasing physical captures during mist-net surveys using sound lures. Because much bat monitoring is now performed using acoustic detection, there is a similar need to increase detection probabilities during acoustic surveys. Ultraviolet (UV) lights anecdotally have been shown to attract insects and thereby attract foraging bats for observational studies and to experimentally provide a food source for WNS-impacted bats before and after hibernation. Therefore, we constructed a field-portable and programmable UV lure device to determine the value of lures for increasing acoustic detection of bats. We tested if the lure device increased both the echolocation passes and feeding activity (feeding buzzes) across a transect of bat detectors. There was an increase in feeding activity around the UV light, with a nuanced, species-specific and positionally dependent effect on echolocation passes received. The UV light lure increased echolocation passes for the eastern red bat (Lasiurus borealis), little brown bat (Myotis lucifugus), and evening bat (Nycticeius humeralis), but decreased passes of the North American hoary bat (Lasiurus cinereus). The northern long-eared bat (Myotis septentrionalis) showed a negative response within the illuminated area but increased echolocation activity outside the illuminated area during lure treatment and activity was elevated at all positions after the lure was deactivated. Our study demonstrates some potential utility of UV lures in increasing the feeding activity and acoustic detection of bats. Additional research and development of UV lure technology may be beneficial, including alternating on and off periods to improve detection of light-averse species, and improving echolocation call quality along with the increase in received passes.

Abstract Mating dynamics can govern species impacts from rapid global change by influencing population rates of growth and adaptation, as well as individual traits that affect mortality risks from novel pressures. Here, we examined sex differences in the activity of Myotis lucifugus during their mating season, which coincides with exposure to the lethal fungal pathogen (Pseudogymnoascus destructans) that causes white‐nose syndrome. We expected differences in activity between the sexes to modify seasonal P. destructans dynamics as the pathogen can replicate only at the cool temperatures at which bats hibernate. We used passive antenna systems installed at the entrances of hibernacula and PIT tags to characterize activity patterns of bats. We also measured pathogen loads on bats during autumn mating and early hibernation to assess how infection changed according to host phenology. We found that females spent fewer days active during autumn, arrived after males, and were primarily active on the warmest nights. In contrast, males were active throughout the entire mating season and later in autumn than females. Importantly, differences in phenology corresponded to higher pathogen loads on females during early hibernation, likely because active males maintained warm body temperatures which inhibited pathogen growth. Differences in activity between sexes and in the transition from swarm to hibernation likely reflect males maximizing their mating opportunities while females conserve energy to meet the cost of spring migration and reproduction. More broadly, our results show how activity during the mating season and phenology can contribute to sex‐biased impacts of a novel disease and highlight the value of understanding species' mating systems to anticipate the impacts of environmental change. Read the free Plain Language Summary for this article on the Journal blog.

The gut microbiome is the community of microbes that inhabits the gastrointestinal tracts of animals. Laboratory findings have shown that the gut microbiome plays a crucial role in host metabolism, physiology, and immunity. This has led to speculation that selection acts on both host and microbiome—although identifying functionally essential coevolving microbes in wild animals remains challenging. A recent surge of studies in wild populations has identified phylogenetic, spatiotemporal, dietary, and social patterns in host-associated microbiomes. Here, we describe and assess the gut microbiomes of two sympatric bat species: big brown bats (Eptesicus fuscus) and little brown bats (Myotis lucifugus). Although these species share similar diets and environments throughout much of their North American ranges, we found they have distinct gut microbiomes. We find no evidence of a functional core microbiome among big brown bats and identify roost identity as a driver of microbiome composition, likely arising from social transmission among hosts through physical proximity. We conclude that both environmental and social factors drive microbiome composition in big brown bats and that repeated, extensive sampling is required to bring ecological reality to host-associated microbiome studies in wild populations.

Bats are reservoir hosts for a number of coronaviruses, some of which may pose spillover risks for humans and other animals. We detected two alphacoronaviruses in big brown bats (Eptesicus fuscus) and little brown myotis (Myotis lucifugus) in Ontario, Canada. These viruses are closely related to other coronaviruses circulating in bats in North America and also distantly related to human and swine coronaviruses. We found high similarity in the receptor-binding domain (RBD) in viruses derived from the same species of bat, but markedly lower in those derived from other species. We also functionally characterized the accessory protein ORF3 finding that ORF3 inhibited both IFNβ production and signaling. Our study provides insights into coronavirus diversity in bats in a previously under-sampled region. This work provides a baseline for in-depth surveillance to characterize the transmission dynamics of endemic coronaviruses in free-ranging wildlife, and for exploring the evolutionary relationships between coronaviruses and their hosts.

Understanding how organisms respond to their environments is challenging, especially due to the complex processes related to metabolism, energetics, and reproduction. Glucocorticoids (GCs) are metabolic hormones that provide insight into internal process and may trigger a variety of behaviors. To understand how changes to the environment influence wildlife, we must ideally measure levels of GCs in wildlife populations that are relatively undisturbed by anthropogenic change or natural disaster. In free-ranging mammals, cortisol is a primary GC hormone and can be measured in fur. Bats are the only mammalian order to have evolved true flight and, in North America, most species of bats are in the family Vespertilionidae. By measuring cortisol levels during energetically expensive periods of an animal's life history, we can document 'baseline levels' related to different life stages, sexes, and ages. We collected fur from hoary (Lasiurus cinereus), silver-haired (Lasionycteris noctivagans), and little-brown Myotis (Myotis lucifugus) bats from southern Alberta and Saskatchewan. Herein we report their baseline levels of fur cortisol, noting interspecific differences between species. We found that bats known to migrate longer distances had higher levels compared to bats that migrate shorter distances, and then hibernate. Interestingly, we found no differences in levels between the reproductive stages for any species. Finally, both silver-haired and hoary bats show a strong difference in fur cortisol levels between adults and juveniles. We suggest that the elevated levels are likely associated with events for mothers during lactation which are then incorporated into the pups' fur while nursing.

Host-parasite relationships can affect the dispersal and transmission of parasites. Myodopsylla insignis (Rothchild, 1903), a bat flea, and Spinturnix americanus (Banks, 1902), a bat wing mite, are two common ectoparasites of the little brown myotis (Myotis lucifugus, Le Conte 1831) that differ in life cycles and time spent on the host. Our goal was to compare how life history traits and host-parasite relationships influence the genetic structure and biogeography of co-infecting ectoparasites using S. americanus mites and M. insignis fleas that feed on Myotis lucifugus bats. Ectoparasites were collected from bats captured at maternity roosts between 2010 and 2017 in Atlantic Canada and sequenced for the cytochrome oxidase c subunit 1 gene. We barcoded 223 S. americanus and 87 M. insignis specimens and examined their genetic diversity, genetic structure, and biogeography. We found evidence of a weak association between geographic distance and sequence divergence between Labrador and Nova Scotia for M. insignis and evidence of regional differentiation between the island of Newfoundland and the mainland for S. americanus, similar to previous findings for M. lucifugus. In terms of biogeography, M. insignis likely underwent historical population expansion, particularly in Labrador, whereas S. americanus may have undergone historical population expansion or selection. Our study highlights how host-parasite relationships are influenced at multiple scales by both host and parasite biology and how an understanding of both host and parasite informs predictions on how these dynamics will be affected by disturbances.

ContextEstimating the distribution of rare species is an important component of conservation. The occurrence of such species is often only known from few presence-only observations and the spatial scales at which they select resources is poorly understood. Scientists often select arbitrary discrete spatial scales for estimating distribution models; however, resource selection follows a hierarchical multiscale process and selection of an incorrect scale may bias model inference and lead to misallocation of resources.ObjectivesOur objectives were to develop a Bayesian latent indicator scale selection (BLISS) model capable of estimating spatial scales of resource selection using small presence-only datasets and apply our model to empirical data characteristic of species of conservation concern.MethodsWe developed a BLISS model using the resource selection function (BLISSRSF) and conducted a simulation study to evaluate its ability to predict spatial scales, predictor coefficients, and proportional probability of use under constraints typical of environmental presence-only data, including small occurrence dataset, variable pseudo-absence sample size, pseudo-absence contamination, and spatial scale autocorrelation. We applied BLISSRSF to estimate winter resource selection for little brown (Myotis lucifugus), northern long-eared (M. septentrionalis), and tricolored (Perimyotis subflavus) bats in the Midwest, USA.ResultsSimulations demonstrated that BLISSRSF accurately estimated model parameters when using 10,000 pseudo-absences under constraints of scale autocorrelation and contamination. BLISSRSF yielded predictive models of winter resource selection for each bat species and high fit with cross-validation datasets.ConclusionsAs broad-scale population declines precipitate, the ability to predict species distributions may be more reliant on presence-only data, particularly for species that are difficult to sample or those which lack adequate support for rigorous field study. BLISSRSF is an adaptable and computationally efficient method for estimating RSF parameters using small presence-only datasets under a used–available study design.

Abstract The ability to identify and track individuals is a critical tool for understanding population demographics and developing effective wildlife management strategies. Forearm bands have been a critical mark–recapture methodology for bats for almost a century despite some concerns that banding may have a negative impact on bat populations. We used banding data from a long-term demographic study of Little Brown Myotis (Myotis lucifugus) to determine rates of band loss and band injury for plastic split-ring (3.5 mm inner diameter), small (2.9 mm) aluminum flanged, and 2 types of large (4.2 mm) aluminum flanged forearm bands (USFWS #2 and Lambourne). We found that bats with plastic split-ring bands and larger aluminum bands (both USFWS #2 and Lambourne) had higher rates of band injuries than bats banded with the smaller (2.9 mm) bands. We also found that the loss rate of the larger 4.2 mm bands was significantly higher than for the smaller bands. Bats recaptured with band-related injuries showed high rates of recovery, with 88% of subsequent recaptures showing no evidence of prior injury. Bats banded when they were young-of-the-year had no detectable difference in band loss rates and lower rates of both band chewing and band-related injury compared to bats initially captured as adults. Our data suggest that the perception of banding having a negative impact on bats such as M. lucifugus is likely due to disturbance related to the banding protocol and not due to injuries or mortality of individual bats. Our data suggest that forearm bands, when properly sized and secured to the wing, continue to be a reliable and low-risk marking method for some bat species.

Bat populations are declining worldwide because of threats such as white-nose syndrome and habitat loss. Multiple-year studies are advantageous for looking at population trends over time and determining the extent of declines. While numerous multiple-year studies have been conducted for bats, they are limited in regional and local coverage. This study compiled the results of 282 walking surveys across 10 years of acoustic bat recording data collected in 3 northwest Ohio parks from 2011 to 2021 to examine temporal trends in bat activity. Overall bat activity (all species combined, average per night) decreased from 2011 to 2018 but showed a slight positive trend from 2019 to 2021. Average activity per night for big brown (Eptesicus fuscus), northern long-eared (Myotis septentrionalis), and little brown bats (Myotis lucifugus) decreased significantly (75%, 95%, and 33% decreases from 2011 to 2021, respectively). Average activity per night of hoary (Lasiurus cinereus) and silver-haired bats (Lasionycteris noctivagans) increased significantly in activity until 2016 (169% and 163% increase from 2011 to 2016, respectively) before decreasing in activity to be closer to initial levels, although their general trend in activity was positive overall (92% and 32% increase from 2011 to 2021, respectively). These data show the advantages of monitoring bat activity over more years to make inferences about population trends. Significant decreases in activity for big brown, little brown, and northern long-eared bats from 2011 to 2021 suggest alterations in bat community structure. These results illustrate a decline in overall bat activity over the last decade. This study also provides an example of volunteer-collected long-term bat monitoring data.

Members of the piggyBac superfamily of DNA transposons are widely distributed in host genomes ranging from insects to mammals. The human genome has retained five piggyBac-derived genes as domesticated elements although they are no longer mobile. Here, we have investigated the transposition properties of piggyBat from Myotis lucifugus, the only known active mammalian DNA transposon, and show that its low activity in human cells is due to subterminal inhibitory DNA sequences. Activity can be dramatically improved by their removal, suggesting the existence of a mechanism for the suppression of transposon activity. The cryo-electron microscopy structure of the piggyBat transposase pre-synaptic complex showed an unexpected mode of DNA binding and recognition using C-terminal domains that are topologically different from those of the piggyBac transposase. Here we show that structure-based rational re-engineering of the transposase through the removal of putative phosphorylation sites and a changed domain organization - in combination with truncated transposon ends - results in a transposition system that is at least 100-fold more active than wild-type piggyBat.

We present the first use of a bioengineered mammalian transposase system derived from Myotis lucifugus (bMLT) for integration of expression vectors into the CHO genome, focusing on GFP and trastuzumab production. Initially, CHO‐K1 cells are transfected with a GFP reporter and varying amounts of bMLT DNA or mRNA. GFP expression is monitored over 17 weeks without selective pressure. Transfection efficiency shows around 90% GFP‐positive cells, but in control cultures GFP expression disappears after 10 days. In contrast, bMLT‐treated cultures maintain stable GFP expression, with a dose‐dependent integration efficiency of up to 60%. The highest GFP expression per cell is observed with lower bMLT amounts. Next‐generation sequencing analysis reveals multiple integration sites, with 85% correctly integrated sequences. Next, CHO‐GS−/− cells are transfected with trastuzumab and bMLT DNA or mRNA. Cells are selected in glutamine‐free medium with varying methionine sulfoximine (MSX) concentrations. Recovery is faster without MSX, and no difference is observed between bMLT DNA and mRNA transfections. bMLT‐treated cultures show a higher percentage of trastuzumab‐secreting cells (40%–55%) compared with random integration (0.3%–0.5%). The absence of insulators in the trastuzumab plasmid likely affects selection behavior, as integration in heterochromatic regions results in gene repression. Overall, bMLT‐mediated integration proves efficient, generating stable cell pools with high expression profiles without selective pressure. The integration sites' genomic location significantly impacts productivity, with favorable regions supporting higher expression. This method shows promise for the rapid and efficient generation of high‐producing cell lines and for rapid evaluation of long‐term effects of different cell engineering approaches.

Rabies continues to affect wildlife and domestic animals in northern Canada and Alaska, but no cases have been reported in the Yukon since the 1970s. To better understand the presence of rabies in the territory, a surveillance program began in 2009, using opportunistically collected samples from 13 species of wild mammals. Samples were collected from trapped wildlife through carcass collection programs, from animals that were found dead by the public, and those involved in human-wildlife conflict or in vehicle collisions. All samples ( n = 763) were negative for rabies virus antigen. Five (<1% of individuals tested) Arctic foxes ( Vulpes lagopus), the primary reservoir of the Arctic rabies strain, were tested. Canada lynx ( Lynx canadensis; n = 290; 38%), wolverine ( Gulo gulo; n = 108; 14%), wolf ( Canis lupus; n = 105; 14%), red fox ( Vulpes vulpes; n = 90; 12%), and little brown bats ( Myotis lucifugus; n = 55; 7%) comprised the majority of animals sampled. Shifts in distributional ranges of species and movements of migratory populations or dispersing individuals, especially from adjacent jurisdictions where rabies is reported, may rapidly alter rabies transmission dynamics in the Yukon. Thus, more targeted surveillance of reservoir species, especially at jurisdictional borders, is recommended to monitor for changes in rabies prevalence in the Yukon.

Abstract White‐nose syndrome is a skin disease of bats caused by the fungus Pseudogymnoascus destructans (Pd). Pd has devastated populations of some bat species in North America, where environmental reservoirs of the fungus are considered a threat to the persistence of bat populations. However, long‐term patterns of Pd environmental persistence in North American hibernacula are unknown. We swabbed hibernacula walls 11 years after the invasion of Pd into Maritime Canada in 2011. This is the first study to examine the persistence of Pd in North American hibernacula >7 years after the first documentation of Pd at a site. The proportion of hibernacula wall swabs with viable Pd decreased over time, with 40.6% of wall swabs positive (n = 32) in 2012, 35.0% (n = 40) in 2015, and 1.7% (n = 120) in 2022. In early winter 2022, 41.18% (n = 17) of bats (Myotis lucifugus, M. septentrionalis, and Perimyotis subflavus) were Pd‐positive compared to 6.67% (n = 15) in late winter, a low prevalence and the opposite pattern compared to the first 4 years after Pd invasion to sites. Our results suggest that Pd loads in the environment naturally decrease to low or undetectable levels over time in our study region. Since attempts to reduce environmental reservoirs have a high likelihood of negative nontarget effects on hibernacula ecosystems, and a low likelihood of completely eradicating Pd, actions to reduce environmental reservoirs in hibernacula should consider deprioritizing sites where Pd has been present >10 years. We urge the collection of further data across hibernacula sites with varied geochemistry, microclimates, organic matter availability, timing of Pd arrival, and surviving bat colony sizes. This will allow a more comprehensive assessment of this strategy.