Infectious diseases of wildlife are increasing worldwide with implications for conservation and human public health. The microbiota (i.e. microbial community living on or in a host) could influence wildlife disease resistance or tolerance. White-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans (Pd), has killed millions of hibernating North American bats since 2007. We characterized the skin microbiota of naïve, pre-WNS little brown bats (Myotis lucifugus) from three WNS-negative hibernation sites and persisting, previously exposed bats from three WNS-positive sites to test the hypothesis that the skin microbiota of bats shifts following WNS invasion. Using high-throughput 16S rRNA gene sequencing on 66 bats and 11 environmental samples, we found that hibernation site strongly influenced the composition and diversity of the skin microbiota. Bats from WNS-positive and WNS-negative sites differed in alpha and beta diversity, as well as in microbiota composition. Alpha diversity was reduced in persisting, WNS-positive bats, and the microbiota profile was enriched with particular taxa such Janthinobacterium, Micrococcaceae, Pseudomonas, Ralstonia, and Rhodococcus. Some of these taxa are recognized for their antifungal activity, and specific strains of Rhodococcus and Pseudomonas are known to inhibit Pd growth. Composition of the microbial community in the hibernaculum environment and the community on bat skin was superficially similar but differed in relative abundance of some bacterial taxa. Our results are consistent with the hypothesis that Pd invasion leads to a shift in the skin microbiota of surviving bats and suggest the possibility that the microbiota plays a protective role for bats facing WNS. The detection of what appears to be enrichment of beneficial bacteria in the skin microbiota of persisting bats is a promising discovery for species re-establishment. Our findings highlight not only the potential value of management actions that might encourage transmission, growth, and establishment of beneficial bacteria on bats, and within hibernacula, but also the potential risks of such management actions.

Background: A wide range of microorganisms inhabit animal skin. This microbial community (microbiome) plays an important role in host defense against pathogens and disease. Bats (Chiroptera: Mammalia) are an ecologically and evolutionarily diversified group with a relatively unexplored skin microbiome. The bat skin microbiome could play a role in disease resistance, for example, to white nose syndrome (WNS), an infection which has been devastating North American bat populations. However, fundamental knowledge of the bat skin microbiome is needed before understanding its role in health and disease resistance. Captive neotropical frugivorous batsArtibeus jamaicensisandCarollia perspicillataprovide a simple controlled system in which to characterize the factors shaping the bat microbiome. Here, we aimed to determine the relative importance of habitat and host species on the bat skin microbiome.Methods: We performed high-throughput 16S rRNA gene sequencing of the skin microbiome of two different bat species living in captivity in two different habitats. In the first habitat,A. jamaicensisandC. perspicillatalived together, while the second habitat contained onlyA. jamaicensis.Results: We found that both habitat and host species shape the composition and diversity of the skin microbiome, with habitat having the strongest influence. CohabitatingA. jamaicensisandC. perspicillatashared more similar skin microbiomes than members of the same species (A. jamaicensis) across two habitats.Discussion: These results suggest that in captivity, the skin microbial community is homogenised by the shared environments and individual proximities of bats living together in the same habitat, at the expense of the innate host species factors. The predominant influence of habitat suggests that environmental microorganisms or pathogens might colonize bat skin. We also propose that bat populations could differ in pathogen susceptibility depending on their immediate environment and habitat.

Little is known about skin microbiota in the context of the disease white-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans (Pd), that has caused enormous declines of hibernating North American bats over the past decade. Interestingly, some hibernating species, such as the big brown bat (Eptesicus fuscus), appear resistant to the disease and their skin microbiota could play a role. However, a comprehensive analysis of the skin microbiota of E. fuscus in the context of Pd has not been done. In January 2017, we captured hibernating E. fuscus, sampled their skin microbiota, and inoculated them with Pd or sham inoculum. We allowed the bats to hibernate in the lab under controlled conditions for 11 weeks and then sampled their skin microbiota to test the following hypotheses: (1) Pd infection would not disrupt the skin microbiota of Pd-resistant E. fuscus; and (2) microbial taxa with antifungal properties would be abundant both before and after inoculation with Pd. Using high-throughput 16S rRNA gene sequencing, we discovered that beta diversity of Pd-inoculated bats changed more over time than that of sham-inoculated bats. Still, the most abundant taxa in the community were stable throughout the experiment. Among the most abundant taxa, Pseudomonas and Rhodococcus are known for antifungal potential against Pd and other fungi. Thus, in contrast to hypothesis 1, Pd infection destabilized the skin microbiota but consistent with hypothesis 2, bacteria with known antifungal properties remained abundant and stable on the skin. This study is the first to provide a comprehensive survey of skin microbiota of E. fuscus, suggesting potential associations between the bat skin microbiota and resistance to the Pd infection and WNS. These results set the stage for future studies to characterize microbiota gene expression, better understand mechanisms of resistance to WNS, and help develop conservation strategies.

Diet has been suggested to be an important driver of variation in microbiota composition in mammals. However, whether this is a more general phenomenon and how fast changes in gut microbiota occur with changes in diet remains poorly understood. Forty-nine years ago, ten lizards of the species Podarcis siculus were taken from the island of Pod Kopište and introduced onto the island of Pod Mrčaru (Croatia). The introduced population underwent a significant dietary shift, and their descendants became omnivorous (consuming up to 80% plant material during summer). Variation in their gut microbiota has never been investigated. To elucidate the possible impact on the gut microbiota of this rapid change in diet, we compared the microbiota (V4 region of the 16S rRNA gene) of P. siculus from Pod Mrčaru, Pod Kopište, and the mainland. In addition, we explored other drivers of variation in gut microbiota including insularity, the population of origin, and the year of sampling. Alpha-diversity analyses showed that the microbial diversity of omnivorous lizards was higher than the microbial diversity of insectivorous lizards. Moreover, omnivorous individuals harbored significantly more Methanobrevibacter. The gut microbial diversity of insectivorous lizards was nonetheless more heterogeneous. Insectivorous lizards on the mainland had different gut microbial communities than their counterparts on the island of Pod Kopište. Bacillus and Desulfovibrio were more abundant in the gut microbiota from insular lizards compared to mainland lizards. Finally, we showed that the population of origin was also an important driver of the composition of the gut microbiota. The dietary shift that occurred in the introduced population of P. siculus has had a detectable impact on the gut microbiota, but other factors such as insularity and the population of origin also contributed to differences in the gut microbial composition of these lizards, illustrating the multifactorial nature of the drivers of variation in gut microbiota. Overall, our data show that changes in gut microbiota may take place on ecological timescales. Yet, diet is only one of many factors driving variation in gut microbiota across populations.

Corrigendum: Antifungal Potential of the Skin Microbiota of Hibernating Big Brown Bats (Eptesicus fuscus) Infected With the Causal Agent of White-Nose Syndrome

The role of the skin microbiome in resistance and susceptibility of wildlife to fungal pathogens has been examined from a taxonomic perspective but skin microbial function, in the context of fungal infection, has yet to be studied. Our objective was to understand effects of a bat fungal pathogen site infection status and course of invasion on skin microbial function. We sampled seven hibernating colonies of Myotis lucifugus covering three-time points over the course of Pseudogymnoascus destructans (Pd) invasion and white nose syndrome (pre-invasion, epidemic, and established). Our results support three new hypotheses about Pd and skin functional microbiome: (1) there is an important effect of Pd invasion stage, especially at the epidemic stage; (2) disruption by the fungus at the epidemic stage could decrease anti-fungal functions with potential negative effects on the microbiome and bat health; (3) the collection site might have a larger influence on microbiomes at the pre-invasion stage rather than at epidemic and established stages. Future studies with larger sample sizes and using meta-omics approaches will help confirm these hypotheses, and determine the influence of the microbiome on wildlife survival to fungal disease.