Whole Genome Sequencing and Phylogenetic Analysis of Rabies Viruses from Bats in Connecticut, USA, 2018–2019

We performed whole genome sequencing and genetic characterization of rabies viruses (RABV) detected in bats submitted to the Connecticut Veterinary Medical Diagnostic Laboratory (CVMDL) during 2018-2019. Among 88 bats submitted to CVMDL, six brain samples (6.8%, 95% confidence interval: 1.6% to 12.1%) tested positive by direct fluorescent antibody test. RABVs were detected in big brown bats (Eptesicus fuscus, n = 4), a hoary bat (Lasiurus cinereus, n = 1), and an unidentified bat species (n = 1). Complete coding sequences of four out of six detected RABVs were obtained. In phylogenetic analysis, the RABVs (18-62, 18-4347, and 19-2274) from big brown bats belong to the bats EF-E1 clade, clustering with RABVs detected from the same bat species in Pennsylvania and New Jersey. The bat RABV (19-2898) detected from the migratory hoary bat belongs to the bats LC clade, clustering with the eleven viruses detected from the same species in Arizona, Washington, Idaho, and Tennessee. The approach used in this study generated novel data regarding genetic relationships of RABV variants, including their reservoirs, and their spatial origin and it would be useful as reference data for future investigations on RABV in North America. Continued surveillance and genome sequencing of bat RABV would be needed to monitor virus evolution and transmission, and to assess the emergence of genetic mutations that may be relevant for public health.

Timing and Weather Offer Alternative Mitigation Strategies for Lowering Bat Mortality at Wind Energy Facilities in Ontario

Relatively high mortality of migratory bats at wind energy facilities has prompted research to understand the underlying spatial and temporal factors, with the goal of developing more effective mitigation approaches. We examined acoustic recordings of echolocation calls at 12 sites and post-construction carcass survey data collected at 10 wind energy facilities in Ontario, Canada, to quantify the degree to which timing and regional-scale weather predict bat activity and mortality. Rain and low temperatures consistently predicted low mortality and activity of big brown bats (Eptesicus fuscus) and three species of migratory tree bats: hoary bat (Lasiurus cinereus), eastern red bat (L. borealis), and silver-haired bat (Lasionycteris noctivagans). Bat activity occurred in waves with distinct peaks through the season; regardless of seasonal timing, most activities occurred in the first half of the night. We conclude that wind energy facilities could adopt a novel and more effective curtailment strategy based on weather and seasonal and nocturnal timing that would minimize mortality risks for bats while increasing the opportunities for power generation, relative to the mitigation strategy of increasing cut-in wind speed to 5.5 m/s.

Impact of Molecular Modifications on the Immunogenicity and Efficacy of Recombinant Raccoon Poxvirus-Vectored Rabies Vaccine Candidates in Mice

Rabies is an ancient disease that is responsible for approximately 59,000 human deaths annually. Bats (Order Chiroptera) are thought to be the original hosts of rabies virus (RABV) and currently account for most rabies cases in wildlife in the Americas. Vaccination is being used to manage rabies in other wildlife reservoirs like fox and raccoon, but no rabies vaccine is available for bats. We previously developed a recombinant raccoonpox virus (RCN) vaccine candidate expressing a mosaic glycoprotein (MoG) gene that protected mice and big brown bats when challenged with RABV. In this study, we developed two new recombinant RCN candidates expressing MoG (RCN-tPA-MoG and RCN-SS-TD-MoG) with the aim of improving RCN-MoG. We assessed and compared in vitro expression, in vivo immunogenicity, and protective efficacy in vaccinated mice challenged intracerebrally with RABV. All three candidates induced significant humoral immune responses, and inoculation with RCN-tPA-MoG or RCN-MoG significantly increased survival after RABV challenge. These results demonstrate the importance of considering molecular elements in the design of vaccines, and that vaccination with either RCN-tPA-MoG or RCN-MoG confers adequate protection from rabies infection, and either may be a sufficient vaccine candidate for bats in future work.

Ixodes scapularis (Ixodida: Ixodidae) Parasitizing an Unlikely Host: Big Brown Bats, Eptesicus fuscus (Chiroptera: Vespertilionidae), in New York State, USA

Ixodes scapularis Say is a three-host tick that has been recorded feeding on over 150 different species of terrestrial vertebrates (mammals, birds, and reptiles). This tick is found throughout the northeastern, coastal southeastern, and upper midwestern United States and is considered the most significant vector of tick-borne pathogens to humans in North America. Despite its ubiquity and broad host range, I. scapularis previously has not been reported feeding on bats (Chiroptera). However, during 2019 and 2020, larvae and nymphs of I. scapularis were recovered from big brown bats, Eptesicus fuscus (Palisot de Beauvois), at four locations in rural New York State, USA. All Ixodes infested bats were injured and found on the ground; therefore, parasitism by I. scapularis was likely opportunistic. Nonetheless, the large number of pathogens known to be associated with bats and the frequency with which I. scapularis bites people suggest that this host–tick relationship is of at least potential epidemiological significance.

Amplitude discrimination is predictably affected by echo frequency filtering in wideband echolocating bats

Big brown bats emit wideband frequency modulated (FM) ultrasonic pulses for echolocation. They perceive target range from echo delay and target size from echo amplitude. Their sounds contain two prominent down-sweeping harmonic sweeps (FM1, ~55-22 kHz; FM2, ~100-55 kHz), which are affected differently by propagation out to the target and back to the bat. FM2 is attenuated more than FM1 during propagation. Bats anchor target ranging asymmetrically on the low frequencies in FM1, while FM2 only contributes if FM1 is present as well. These experiments tested whether the bat's ability to discriminate target size from the amplitude of echoes is affected by selectively attenuating upper or lower frequencies. Bats were trained to perform an echo amplitude discrimination task with virtual echo targets 83 cm away. While echo delay was held constant and echo amplitude was varied to estimate threshold, either lower FM1 frequencies or higher FM2 frequencies were attenuated. The results parallel effects seen in echo delay experiments; bats' performance was significantly poorer when the lower frequencies in echoes were attenuated, compared to higher frequencies. The bat's ability to distinguish between virtual targets at the same simulated range from echoes arriving at the same delay indicates a high level of focused attention for perceptual isolation of one and suppression of the other.

Humidity, huddling & the hibernation energetics of big brown bats (Eptesicus fuscus)

During winter, many mammals hibernate and lower their body temperature and metabolic rate (MR) in prolonged periods of torpor. Hibernators will use energetically expensive arousals (i.e., restore body temperature and MR) presumably to re-establish water balance. Some hibernating mammals however will huddle in groups, possibly to decrease energetic costs and total evaporative water loss (EWL), although the benefit is not fully understood. Research on the relationship between behaviour, physiology, water loss, and energy expenditure of bats during hibernation is especially important because of a fungal disease called white-nose syndrome (WNS). To date, 12 North American bat species are affected by WNS, however big brown bats (Eptesicus fuscus) appear resistant, although the underlying mechanism is poorly understood. The overall objective of my thesis was to understand the influence of humidity and huddling on the behavioural and physiological responses of hibernating big brown bats. To test my hypotheses, I used a captive colony of hibernating big brown bats (n = 20). Specifically, for Chapter 2, I first tested the hypothesis that big brown bats adjust huddling and drinking behaviour depending on humidity, to maintain a consistent pattern of periodic arousals, and therefore energy balance during hibernation. I found that bats hibernating in a dry environment did not differ in arousal/torpor bout frequency, or torpor bout duration throughout hibernation but drank at twice the rate as bats in a humid environment. Bats in the dry treatment also had shorter arousals, and huddled in a denser huddle, potentially to reduce rates of total EWL. During late hibernation, for Chapter 3, I used open-flow respirometry to test two additional hypotheses, first that phenotypic flexibility in total EWL helps explain the tolerance of hibernating big brown bats for a wide range of humidity relative to other bat species. I found that dry-acclimated bats had lower rates of total EWL, compared to bats acclimated to humid conditions. I then tested the second hypothesis that big brown bats can use huddling to mitigate the challenge of dry conditions. I found that, for humid-acclimated bats, rates of total EWL were reduced with huddling bats but there was no effect of huddling on EWL for bats acclimated to dry conditions. These results suggest that the ability of big brown bats to reduce rates of total EWL through acclimation may reduce the need to huddle with conspecifics to avoid water loss and thus dehydration. Overall, my thesis suggests that big brown bats use both behavioural and physiological mechanisms to reduce water loss which could allow them to exploit habitats for hibernation that are unavailable to other bat species and could also help explain their apparent resistance to WNS.

Evaluating odour and urinary sex preferences in the big brown bat (Eptesicus fuscus)

Olfactory cues provide detailed information to mammals regarding conspecifics. Bats may identify species, colony membership, and individual’s using olfaction. Big brown bats (Eptesicus fuscus (Palisot de Beauvois, 1976)) live in mixed-sex colonies and must differentiate between sexes to locate mates. We hypothesized that odour cues convey information about sex. In Experiment 1, adult E. fuscus were recorded exploring a Y-maze that contained general body odours sampled from male or female conspecifics. One group of subjects was habituated to the Y-maze prior to experimental trials, whereas a second group was not. Bat exploration and the proportion of time spent near each scent were used as preference indicators for the body odour of a particular sex. Experiment 2 followed similar procedures except the odour cue tested was urine from either male or female conspecifics and without Y-maze habituation. Results found no evidence that E. fuscus prefer the body odours of a given sex, but females did prefer the odour of male urine. Non-habituated animals in Experiment 1 were more likely to explore the Y-maze and approach a stimulus scent compared to habituated bats. These findings have important implications for courtship and mating behaviour in bats, as well for designing future behavioural studies.

Hepatic lipid signatures of little brown bats (Myotis lucifugus) and big brown bats (Eptesicus fuscus) at early stages of white-nose syndrome

White-nose syndrome (WNS) is an emergent wildlife fungal disease of cave-dwelling, hibernating bats that has led to unprecedented mortalities throughout North America. A primary factor in WNS-associated bat mortality includes increased arousals from torpor and premature fat depletion during winter months. Details of species and sex-specific changes in lipid metabolism during WNS are poorly understood and may play an important role in the pathophysiology of the disease. Given the likely role of fat metabolism in WNS and the fact that the liver plays a crucial role in fatty acid distribution and lipid storage, we assessed hepatic lipid signatures of little brown bats (Myotis lucifugus) and big brown bats (Eptesicus fuscus) at an early stage of infection with the etiological agent, Pseudogymnoascus destructans (Pd). Differences in lipid profiles were detected at the species and sex level in the sham-inoculated treatment, most strikingly in higher hepatic triacylglyceride (TG) levels in E. fuscus females compared to males. Interestingly, several dominant TGs (storage lipids) decreased dramatically after Pd infection in both female M. lucifugus and E. fuscus. Increases in hepatic glycerophospholipid (structural lipid) levels were only observed in M. lucifugus, including two phosphatidylcholines (PC [32:1], PC [42:6]) and one phosphatidylglycerol (PG [34:1]). These results suggest that even at early stages of WNS, changes in hepatic lipid mobilization may occur and be species and sex specific. As pre-hibernation lipid reserves may aid in bat persistence and survival during WNS, these early perturbations to lipid metabolism could have important implications for management responses that aid in pre-hibernation fat storage.

Bionic target localization method based on range-azimuth joint dictionary

The traditional underwater sonar system usually achieve high angle resolution by increasing array aperture and the number of array elements, but this method will inevitably lead to complex system and high cost. Given that big brown bats have obtained surprisingly high resolution using a simple system, this paper proposes a bionic target localization method. First, a range-azimuth joint dictionary was constructed based on the bionic system of multi-harmonic emission and double random array reception. Then, the coherence characteristic of the dictionary was analyzed and the range and azimuth of the target were estimated, and at last the experimental verification was completed. The results show that the bionic range-azimuth joint estimation based on sparse signal representation can achieve high-precision target localization under the condition of echo high aliasing.