Microbiota diversity and anti-Pseudogymnoascus destructans bacteria isolated from Myotis pilosus skin during late hibernation.

Symbiotic microorganisms that reside on the host skin serve as the primary defense against pathogens in vertebrates. Specifically, the skin microbiome of bats may play a crucial role in providing resistance against Pseudogymnoascus destructans (Pd), the pathogen causing white-nose syndrome. However, the epidermis symbiotic microbiome and its specific role in resisting Pd in highly resistant bats in Asia are still not well understood. In this study, we collected and characterized skin microbiota samples of 19 Myotis pilosus in China and explored the differences between Pd-positive and negative individuals. We identified inhibitory effects of these bacteria through cultivation methods. Our results revealed that the Simpson diversity index of the skin microbiota for positive individuals was significantly lower than that of negative individuals, and the relative abundance of Pseudomonas was significantly higher in positive bats. Regardless of whether individuals were positive or negative for Pd, the relative abundance of potentially antifungal genera in skin microbiota was high. Moreover, we successfully isolated 165 microbes from bat skin and 41 isolates from positive individuals able to inhibit Pd growth compared to only 12 isolates from negative individuals. A total of 10 genera of Pd-inhibiting bacteria were screened, among which the genera Algoriella, Glutamicibacter, and Psychrobacter were newly discovered as Pd-inhibiting genera. These Pd-inhibiting bacteria metabolized a variety of volatile compounds, including dimethyl trisulfide, dimethyl disulfide, propylene sulfide, 2-undecanone, and 2-nonanone, which were able to completely inhibit Pd growth at low concentrations.IMPORTANCERecently, white-nose syndrome has caused the deaths of millions of hibernating bats, even threatening some with regional extinction. Bats in China with high resistance to Pseudogymnoascus destructans can provide a powerful reference for studying the management of white-nose syndrome and understanding the bats against the pathogen's intrinsic mechanisms. This study sheds light on the crucial role of host symbiotic skin microorganisms in resistance to pathogenic fungi and highlights the potential for harnessing natural defense mechanisms for the prevention and treatment of white-nose syndrome. In addition, this may also provide promising candidates for the development of bioinsecticides and fungicides that offer new avenues for addressing fungal diseases in wildlife and agricultural environments.

A Palearctic view of a bat fungal disease.

The fungal infection causing white-nose disease in hibernating bats in North America has resulted in dramatic population declines of affected species, since the introduction of the causative agent Pseudogymnoascus destructans. The fungus is native to the Palearctic, where it also infects several bat species, yet rarely causes severe pathology or the death of the host. Pseudogymnoascus destructans infects bats during hibernation by invading and digesting the skin tissue, resulting in the disruption of torpor patterns and consequent emaciation. Relations among pathogen, host, and environment are complex, and individuals, populations, and species respond to the fungal pathogen in different ways. For example, the Nearctic Myotis lucifugus responds to infection by mounting a robust immune response, leading to immunopathology often contributing to mortality. In contrast, the Palearctic M. myotis shows no significant immunological response to infection. This lack of a strong response, resulting from the long coevolution between the hosts and the pathogen in the pathogen's native range, likely contributes to survival in tolerant species. After more than 15 years since the initial introduction of the fungus to North America, some of the affected populations are showing signs of recovery, suggesting that the fungus, hosts, or both are undergoing processes that may eventually lead to coexistence. The suggested or implemented management methods of the disease in North America have encompassed, for example, the use of probiotics and fungicides, vaccinations, and modifying the environmental conditions of the hibernation sites to limit the growth of the pathogen, intensity of infection, or the hosts' responses to it. Based on current knowledge from Eurasia, policy makers and conservation managers should refrain from disrupting the ongoing evolutionary processes and adopt a holistic approach to managing the epizootic.

Vista paleártica de una enfermedad fúngica de murciélagos Resumen La enfermedad fúngica que produce el síndrome de nariz blanca en murciélagos en hibernación en Norte América ha resultado en declinaciones poblacionales dramáticas en las especies afectadas desde la introducción del agente causante, Pseudogymnoascus destructans. El hongo es nativo del Paleártico, donde también infecta a varias especies de murciélagos; sin embargo, raramente causa patología severa o la muerte del hospedero. Pseudogymnoascus destructans infecta a los murciélagos durante la hibernación invadiendo y digiriendo el tejido de la piel, lo que resulta en la disrupción de los patrones de torpor y la consecuente emaciación. Las relaciones entre el patógeno, el huésped y el ambiente son complejas, y los individuos, las especies y poblaciones responden al patógeno fúngico de distintas maneras. Por ejemplo, Myotis lucifugus, especie del Neártico, responde a la infección montando una respuesta inmune robusta, produciendo una inmunopatología que a menudo contribuye a la mortalidad. En contraste, M. myotis del Paleártico no presenta respuesta inmunológica significativa a la infección. La falta de una fuerte respuesta, resultado de la larga coevolución entre hospederos y el patógeno en el rango nativo de distribución del patógeno, probablemente contribuye a la supervivencia en especies tolerantes. Después de más de 15 años desde la introducción del hongo en Norte América, algunas de las poblaciones afectadas están mostrando señales recuperación, lo que sugiere que el hongo, hospederos, o ambos, están pasando por procesos que eventualmente pueden conducir a la coexistencia. Los métodos de manejo de la enfermedad sugeridos o implementados en Norte América han abarcado, por ejemplo, el uso de probióticos y fungicidas, vacunaciones y modificación de las condiciones ambientales de los sitios de hibernación para limitar el crecimiento del patógeno, la intensidad de la infección o las respuestas de los hospederos. Con base en conocimiento actual de Eurasia, los formuladores de políticas y los manejadores de la conservación deberían abstenerse de alterar los procesos evolutivos en curso y adoptar un enfoque holístico para gestionar la epizootia.

Validation of a Field-Portable, Handheld Real-Time PCR System for Detecting Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome in Bats.

White-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans, has decimated bat populations across North America. Despite ongoing management programs, WNS continues to expand into new populations, including in US states previously thought to be free from the pathogen and disease. This expansion highlights a growing need for surveillance tools that can be used to enhance existing monitoring programs and support the early detection of P. destructans in new areas. We evaluated the feasibility of using a handheld, field-portable, real-time (quantitative) PCR (qPCR) thermocycler known as the Biomeme two3 and the associated field-based nucleic acid extraction kit and assay reagents for the detection of P. destructans in little brown bats (Myotis lucifugus). Results from the field-based protocol using the Biomeme platform were compared with those from a commonly used laboratory-based qPCR protocol. When using dilutions of known conidia concentrations, the lowest detectable concentration with the laboratory-based approach was 108.8 conidia/mL, compared with 1,087.5 conidia/mL (10 times higher, i.e., one fewer 10× dilution) using the field-based approach. Further comparisons using field samples suggest a high level of concordance between the two protocols, with positive and negative agreements of 98.2% and 100% respectively. The cycle threshold values were marginally higher for most samples using the field-based protocol. These results are an important step in establishing and validating a rapid, field-assessable detection platform for P. destructans, which is urgently needed to improve the surveillance and monitoring capacity for WNS and support on-the-ground management and response efforts.

Capture rates of Eptesicus fuscus increase following white-nose syndrome across the eastern US.

Emerging infectious diseases threaten wildlife globally. While the effects of infectious diseases on hosts with severe infections and high mortality rates often receive considerable attention, effects on hosts that persist despite infection are less frequently studied. To understand how persisting host populations change in the face of disease, we quantified changes to the capture rates of Eptesicus fuscus (big brown bats), a persisting species susceptible to infection by the invasive fungal pathogen Pseudogymnoascus destructans (Pd; causative agent for white-nose syndrome), across the eastern US using a 30-year dataset. Capture rates of male and female E. fuscus increased from preinvasion to pathogen establishment years, with greater increases to the capture rates of females than males. Among females, capture rates of pregnant and post-lactating females increased by pathogen establishment. We outline potential mechanisms for these broad demographic changes in E. fuscus capture rates (i.e., increases to foraging from energy deficits created by Pd infection, increases to relative abundance, or changes to reproductive cycles), and suggest future research for identifying mechanisms for increasing capture rates across the eastern US. These data highlight the importance of understanding how populations of persisting host species change following pathogen invasion across a broad spatial scale. Understanding changes to population composition following pathogen invasion can identify broad ecological patterns across space and time, and open new avenues for research to identify drivers of those patterns.

Bat white-nose disease fungus diversity in time and space.

White-nose disease (WND), caused by the psychrophilic fungus Pseudogymnoascusdestructans, represents one of the greatest threats for North American hibernating bats. Research on molecular data has significantly advanced our knowledge of various aspects of the disease, yet more studies are needed regarding patterns of P.destructans genetic diversity distribution. In the present study, we investigate three sites within the native range of the fungus in detail: two natural hibernacula (karst caves) in Bulgaria, south-eastern Europe and one artificial hibernaculum (disused cellar) in Germany, northern Europe, where we conducted intensive surveys between 2014 and 2019. Using 18 microsatellite and two mating type markers, we describe how P.destructans genetic diversity is distributed between and within sites, the latter including differentiation across years and seasons of sampling; across sampling locations within the site; and between bats and hibernaculum walls. We found significant genetic differentiation between hibernacula, but we could not detect any significant differentiation within hibernacula, based on the variables examined. This indicates that most of the pathogen's movement occurs within sites. Genotypic richness of P.destructans varied between sites within the same order of magnitude, being approximately two times higher in the natural caves (Bulgaria) compared to the disused cellar (Germany). Within all sites, the pathogen's genotypic richness was higher in samples collected from hibernaculum walls than in samples collected from bats, which corresponds with the hypothesis that hibernacula walls represent the environmental reservoir of the fungus. Multiple pathogen genotypes were commonly isolated from a single bat (i.e. from the same swab sample) in all study sites, which might be important to consider when studying disease progression.

When the host's away, the pathogen will play: the protective role of the skin microbiome during hibernation.

The skin of animals is enveloped by a symbiotic microscopic ecosystem known as the microbiome. The host and microbiome exhibit a mutualistic relationship, collectively forming a single evolutionary unit sometimes referred to as a holobiont. Although the holobiome theory highlights the importance of the microbiome, little is known about how the skin microbiome contributes to protecting the host. Existing studies focus on humans or captive animals, but research in wild animals is in its infancy. Specifically, the protective role of the skin microbiome in hibernating animals remains almost entirely overlooked. This is surprising, considering the massive population declines in hibernating North American bats caused by the fungal pathogen Pseudogymnoascus destructans, which causes white-nose syndrome. Hibernation offers a unique setting in which to study the function of the microbiome because, during torpor, the host's immune system becomes suppressed, making it susceptible to infection. We conducted a systematic review of peer-reviewed literature on the protective role of the skin microbiome in non-human animals. We selected 230 publications that mentioned pathogen inhibition by microbes residing on the skin of the host animal. We found that the majority of studies were conducted in North America and focused on the bacterial microbiome of amphibians infected by the chytrid fungus. Despite mentioning pathogen inhibition by the skin microbiome, only 30.4% of studies experimentally tested the actual antimicrobial activity of symbionts. Additionally, only 7.8% of all publications studied defensive cutaneous symbionts during hibernation. With this review, we want to highlight the knowledge gap surrounding skin microbiome research in hibernating animals. For instance, research looking to mitigate the effects of white-nose syndrome in bats should focus on the antifungal microbiome of Palearctic bats, as they survive exposure to the Pseudogymnoascus destructans -pathogen during hibernation. We also recommend future studies prioritize lesser-known microbial symbionts, such as fungi, and investigate the effects of a combination of anti-pathogen microbes, as both areas of research show promise as probiotic treatments. By incorporating the protective skin microbiome into disease mitigation strategies, conservation efforts can be made more effective.