Masting promotes transformation from predation to mutualism in an oak-weevil-rodent system.

The significance of ecological non-monotonicity (a function whose first derivative changes signs) in shaping the structure and functions of the ecosystem has recently been recognized, but such studies involving high-order interactions are rare. Here, we have proposed a three-trophic conceptual diagram on interactions among trees, rodents, and insects in mast and non-mast years and tested the hypothesis that oak (Quercus wutaishanica) masting could result in increased mutualism and less predation in an oak-weevil-rodent system in a warm temperate forest of China. Our 14-year dataset revealed that mast years coincided with a relatively low rodent abundance but a high weevil abundance. Masting not only benefited seedling recruitment of oaks through increased dispersal by rodents but also a decrease in predation by rodents and weevils, as well as an increase in the overwintering survival of rodents. Masting appeared to have increased weevil survival by reducing predation of infested acorns by rodents. These results suggest that masting benefits all participants in the plant-insect-rodent system by increasing mutualism and reducing predation behavior (i.e., a non-monotonic function). Our study highlights the significance of masting in maintaining the diversity and function of the forest ecosystem by facilitating the transformation from predation to mutualism among trophic species.

The role of host traits and geography in shaping the gut microbiome of insectivorous bats.

The gut microbiome is a symbiotic microbial community associated with the host and plays multiple important roles in host physiology, nutrition, and health. A number of factors have been shown to influence the gut microbiome, among which diet is considered to be one of the most important; however, the relationship between diet composition and gut microbiota in wild mammals is still not well recognized. Herein, we characterized the gut microbiota of bats and examined the effects of diet, host taxa, body size, gender, elevation, and latitude on the gut microbiota. The cytochrome C oxidase subunit I (COI) gene and 16S rRNA gene amplicons were sequenced from the feces of eight insectivorous bat species in southern China, including Miniopterus fuliginosus, Aselliscus stoliczkanus, Myotis laniger, Rhinolophus episcopus, Rhinolophus osgoodi, Rhinolophus ferrumequinum, Rhinolophus affinis, and Rhinolophus pusillus. The results showed that the composition of gut microbiome and diet exhibited significant differences among bat species. Diet composition and gut microbiota were significantly correlated at the order, family, genus, and operational taxonomic unit levels, while certain insects had a marked effect on the gut microbiome at specific taxonomic levels. In addition, elevation, latitude, body weight of bats, and host species had significant effects on the gut microbiome, but phylosymbiosis between host phylogeny and gut microbiome was lacking. These findings clarify the relationship between gut microbiome and diet and contribute to improving our understanding of host ecology and the evolution of the gut microbiome in wild mammals. IMPORTANCE: The gut microbiome is critical for the adaptation of wildlife to the dynamic environment. Bats are the second-largest group of mammals with short intestinal tract, yet their gut microbiome is still poorly studied. Herein, we explored the relationships between gut microbiome and food composition, host taxa, body size, gender, elevation, and latitude. We found a significant association between diet composition and gut microbiome in insectivorous bats, with certain insect species having major impacts on gut microbiome. Factors like species taxa, body weight, elevation, and latitude also affected the gut microbiome, but we failed to detect phylosymbiosis between the host phylogeny and the gut microbiome. Overall, our study presents novel insights into how multiple factors shape the bat's gut microbiome together and provides a study case on host-microbe interactions in wildlife.

Seasonal assembly of skin microbiota driven by neutral and selective processes in the greater horseshoe bat.

Skin microbiota play an important role in protecting bat hosts from the fungal pathogen Pseudogymnoascus destructans, which has caused dramatic bat population declines and extinctions. Recent studies have provided insights into the bacterial communities of bat skin, but variation in skin bacterial community structure in the context of the seasonal dynamics of fungal invasion, as well as the processes that drive such variation, remain largely unexplored. In this study, we characterized bat skin microbiota over the course of the bat hibernation and active season stages and used a neutral model of community ecology to determine the relative roles of neutral and selective processes in driving microbial community variation. Our results showed significant seasonal shifts in skin community structure, as well as less diverse microbiota in hibernation than in the active season. Skin microbiota were influenced by the environmental bacterial reservoir. During both the hibernation and active season stages, more than 78% of ASVs in bat skin microbiota were consistent with neutral distribution, implying that neutral processes, that is, dispersal or ecological drift contributing the most to shifts in skin microbiota. In addition, the neutral model showed that some ASVs were actively selected by the bats from the environmental bacterial reservoir, accounting for approximately 20% and 31% of the total community during hibernation and active season stages, respectively. Overall, this research provides insights into the assemblage of bat-associated bacterial communities and will aid in the development of conservation strategies against fungal disease.

Diversifying selection and climatic effects on major histocompatibility complex class II gene diversity in the greater horseshoe bat.

Heterogeneous pathogenic stress can shape major histocompatibility complex (MHC) diversity by influencing the functional plasticity of the immune response. Therefore, MHC diversity could reflect environmental stress, demonstrating its importance in uncovering the mechanisms of adaptive genetic variation. In this study, we combined neutral microsatellite loci, an immune-related MHC II-DRB locus, and climatic factors to unravel the mechanisms affecting the diversity and genetic differentiation of MHC genes in the greater horseshoe bat (Rhinolophus ferrumequinum), a species with a wide geographical distribution that has three distinct genetic lineages in China. First, increased genetic differentiation at the MHC locus among populations compared using microsatellites indicated diversifying selection. Second, the genetic differentiation of MHC and microsatellites were significantly correlated, suggesting that demographic processes exist. However, MHC genetic differentiation was significantly correlated with geographical distance among populations, even after controlling for the neutral markers, suggesting a major effect of selection. Third, although the MHC genetic differentiation was larger than that for microsatellites, there was no significant difference in the genetic differentiation between the two markers among genetic lineages, indicating the effect of balancing selection. Fourth, combined with climatic factors, MHC diversity and supertypes showed significant correlations with temperature and precipitation, but not with the phylogeographic structure of R. ferrumequinum, suggesting an effect of local adaptation driven by climate on MHC diversity. Moreover, the number of MHC supertypes varied between populations and lineages, suggesting regional characteristics and support for local adaptation. Taken together, the results of our study provide insights into the adaptive evolutionary driving forces at different geographic scales in R. ferrumequinum. In addition, climate factors may have played a vital role in driving adaptive evolution in this species.

Matrine alleviates depressive-like behaviors via modulating microbiota-gut-brain axis in CUMS-induced mice.

BACKGROUND: The realization of the "microbiota-gut-brain" axis plays a critical role in neuropsychiatric disorders, particularly depression, is advancing rapidly. Matrine is a natural bioactive compound, which has been found to possess potential antidepressant effect. However, the underlying mechanisms of regulation of the "microbiota-gut-brain" axis in the treatment of depression by oral matrine remain elusive. METHODS: Its antidepressant effects were initially evaluated by behavioral tests and relative levels of monoamine neurotransmitters, and matrine has been observed to attenuate the depression-like behavior and increase neurotransmitter content in CUMS-induced mice. Subsequently, studies from the "gut" to "brain" were conducted, including detection of the composition of gut microbiota by 16S rRNA sequencing; the metabolomics detection of gut metabolites and the analysis of differential metabolic pathways; the assessment of relative levels of diamine oxidase, lipopolysaccharide, pro-inflammatory cytokines, and brain-derived neurotrophic factor (BDNF) by ELISA kits or immunofluorescence. RESULTS: Matrine could regulate the disturbance of gut microbiota and metabolites, restore intestinal permeability, and reduce intestinal inflammation, thereby reducing the levels of pro-inflammatory cytokines in peripheral blood circulation and brain regions, and ultimately increase the levels of BDNF in brain. CONCLUSION: Matrine may ameliorate CUMS-induced depression in mice by modulating the "microbiota-gut-brain" axis.

Temporal dynamics of the bat wing transcriptome: Insight into gene-expression changes that enable protection against pathogen.

Skin acts as a mechanical barrier between the body and its surrounding environment and plays an important role in resistance to pathogens. However, we still know little regarding skin responses to physiological changes, particularly with regard to responses against potential pathogens. We herein executed RNA-seq on the wing of the Rhinolophus ferrumequinum to assess gene-expression variations at four physiological stages: pre-hibernation, hibernation (early-hibernation and late-hibernation), and post-hibernation, as well as the gene-expression patterns of infected and uninfected bats with the Pseudogymnoascus destructans (Pd). Our results showed that a greater number of differentially expressed genes between the more disparate physiological stages. Functional enrichment analysis showed that the down-regulated response pathways in hibernating bats included phosphorus metabolism and immune response, indicating metabolic suppression and decreased whole immune function. We also found up-regulated genes in post-hibernating bats that included C-type lectin receptor signalling, Toll-like receptor signalling pathway, and cell adhesion, suggesting that the immune response and skin integrity of the wing were improved after bats emerged from their hibernation and that this facilitated clearing Pd from the integument. Additionally, we found that the genes involved in cytokine or chemokine activity were up-regulated in late-hibernation compared to early-hibernation and that FOSB regulation of immune cell activation was differentially expressed in bats infected with Pd during late-hibernation, implying that the host's innate immune function was enhanced during late-hibernation so as to resist pathogenic infection. Our findings highlight the concept that maintenance of intrinsic immunity provides protection against pathogenic infections in highly resistant bats.

Extensive Adaptive Variation in Gene Expression within and between Closely Related Horseshoe Bats (Chiroptera, Rhinolophus) Revealed by Three Organs.

In the process of species differentiation and adaption, the relative influence of natural selection on gene expression variation often remains unclear (especially its impact on phenotypic divergence). In this study, we used differentially expressed genes from brain, cochlea, and liver samples collected from two species of bats to determine the gene expression variation forced by natural selection when comparing at the interspecific (Rhinolophus siamensis and R. episcopus episcopus) and the intraspecific (R. e. episcopus and R. episcopus spp.) levels. In both cases, gene expression variation was extensively adaptive (>66.0%) and mainly governed by directional selection, followed by stabilizing selection, and finally balancing selection. The expression variation related to acoustic signals (resting frequency, RF) and body size (forearm length, FA) was also widely governed by natural selection (>69.1%). Different functional patterns of RF- or FA-related adaptive expression variation were found between the two comparisons, which manifested as abundant immune-related regulations between subspecies (indicating a relationship between immune response and phenotypic adaption). Our study verifies the extensive adaptive expression variation between both species and subspecies and provides insight into the effects of natural selection on species differentiation and adaptation as well as phenotypic divergence at the expression level.

Skin Microbiota Variation Among Bat Species in China and Their Potential Defense Against Pathogens.

Host-associated skin bacteria are essential for resisting pathogen infections and maintaining health. However, we have little understanding of how chiropteran skin microbiota are distributed among bat species and their habitats, or of their putative roles in defending against Pseudogymnoascus destructans in China. In this study, we characterized the skin microbiomes of four bat species at five localities using 16S rRNA gene amplicon sequencing to understand their skin microbial composition, structure, and putative relationship with disease. The alpha- and beta-diversities of skin microbiota differed significantly among the bat species, and the differences were affected by environmental temperature, sampling sites, and host body condition. The chiropteran skin microbial communities were enriched in bacterial taxa that had low relative abundances in the environment. Most of the potential functions of skin microbiota in bat species were associated with metabolism. Focusing on their functions of defense against pathogens, we found that skin microbiota could metabolize a variety of active substances that could be potentially used to fight P. destructans. The skin microbial communities of bats in China are related to the environment and the bat host, and may be involved in the host's defense against pathogens.

Bacterial community dynamics on bats and the implications for pathogen resistance.

The bats skin microbiota plays an important role in reducing pathogen infection, including the deadly fungal pathogen Pseudogymnoascus destructans, the causative agent of white-nose syndrome. However, the dynamic of skin bacterial communities response to environmental perturbations remains poorly described. We characterized skin bacterial community over time and space in Rhinolophus ferrumequinum, a species with high resistance to the infection with P. destructans. We collected environmental covariate data to determine what factors influenced changes in community structure. We observed significant temporal and spatial shifts in the skin bacterial community, which was mainly associated with variation in operational taxonomic units. The skin bacterial community differed by the environmental microbial reservoirs and was most influenced by host body condition, bat roosting temperature and geographic distance between sites, but was not influenced by pathogen infection. Furthermore, the skin microbiota was enriched in particular taxa with antifungal abilities, such as Enterococcus, Burkholderia, Flavobacterium, Pseudomonas, Corynebacterium and Rhodococcus. And specific strains of Pseudomonas, Corynebacterium and Rhodococcus even inhibited P. destructans growth. Our findings provide new insights in characterizing the variation in bacterial communities can inform us about the processes of driving community assembly and predict the host's ability to resist or survive pathogen infection.

Activity of bacteria isolated from bats against Pseudogymnoascus destructans in China.

White-nose syndrome, a disease that is caused by the psychrophilic fungus Pseudogymnoascus destructans, has threatened several North America bat species with extinction. Recent studies have shown that East Asian bats are infected with P. destructans but show greatly reduced infections. While several factors have been found to contribute to these reduced infections, the role of specific microbes in limiting P. destructans growth remains unexplored. We isolated three bacterial strains with the ability to inhibit P. destructans, namely, Pseudomonas yamanorum GZD14026, Pseudomonas brenneri XRD11711 and Pseudomonas fragi GZD14479, from bats in China. Pseudomonas yamanorum, with the highest inhibition score, was selected to extract antifungal active substance. Combining mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy analyses, we identified the active compound inhibiting P. destructans as phenazine-1-carboxylic acid (PCA), and the minimal inhibitory concentration (MIC) was 50.12 µg ml-1 . Whole genome sequencing also revealed the existence of PCA biosynthesis gene clusters. Gas chromatography-mass spectrometry (GC-MS) analysis identified volatile organic compounds. The results indicated that 10 ppm octanoic acid, 100 ppm 3-tert-butyl-4-hydroxyanisole (isoprenol) and 100 ppm 3-methyl-3-buten-1-ol (BHA) inhibited the growth of P. destructans. These results support that bacteria may play a role in limiting the growth of P. destructans on bats.

Beneficial Effect of Alkaloids From Sophora alopecuroides L. on CUMS-Induced Depression Model Mice via Modulating Gut Microbiota.

It was recently shown that the gut microbiota of both depression patients and depression model animals is significantly altered, suggesting that gut microbes are closely related to depression. Here, we investigated the effects of Sophora alopecuroides L.-derived alkaloids on the gut microbiota of mice with depression-like behaviors. We first established a mouse model of depression via chronic unpredictable mild stress (CUMS) and detected changes in depression-like behaviors and depression-related indicators. Simultaneously, 16S rRNA sequencing was performed to investigate gut microbiota changes. Sophora alopecuroides L.-derived alkaloids improved depression-like behaviors and depression-related indicators in mice. The alkaloids decreased the gut microbiota diversity of CUMS mice and depleted intestinal differentially abundant "harmful" microbiota genera. Spearman analysis showed that there is a certain correlation between the differential microbiota (Lactobacillus, Helicobacter, Oscillospira, Odoribacter, Mucispirillum, Ruminococcus), depression-like behaviors, and depression-related indicators. Combined with the predictive analysis of gut microbiota function, these results indicate that alkaloids improve depression in mice through modulating gut microbiota.

Changes in the gut microbiota during Asian particolored bat (Vespertilio sinensis) development.

BACKGROUND: The gut microbiota is closely linked to host development, diet and health and is influenced by both the host and the environment. Although many studies have focused on the dynamics of the gut microbiota during development in captive animals, few studies have focused on the dynamics of the gut microbiota during development in wild animals, especially for the order Chiroptera. METHODS: In this study, we characterized the gut microbiota of the wild Asian particolored bat (Vespertilio sinensis) from 1 day to 6 weeks after birth. We explored the changes in their gut microbial community compositions, examined possible influencing factors, and predicted the feeding transition period. RESULTS: The gut microbiota changed during the development of V. sinensis. The alpha diversity of the bats' gut microbiota gradually increased but did not change significantly from the 1st day to the 4th week after birth; however, the alpha diversity decreased significantly in week 5, then stabilized. The beta diversity differed slightly in weeks 4-6. In week 4, the fecal samples showed the highest diversity in bacterial community composition. Thus, we predicted that the potential feeding transition period for V. sinensis may occur during week 4. Redundancy analysis showed that age and body mass index significantly affected the compositional changes of the gut microbiota in Asian particolored bats. CONCLUSION: The gut microbiota changed during the development of V. sinensis. We suggest that changes in the alpha and beta diversity during week 4 after birth indicate a potential feeding transition, highlighting the importance of diet in the gut microbiota during the development of V. sinensis.

Environmental reservoir dynamics predict global infection patterns and population impacts for the fungal disease white-nose syndrome.

Disease outbreaks and pathogen introductions can have significant effects on host populations, and the ability of pathogens to persist in the environment can exacerbate disease impacts by fueling sustained transmission, seasonal epidemics, and repeated spillover events. While theory suggests that the presence of an environmental reservoir increases the risk of host declines and threat of extinction, the influence of reservoir dynamics on transmission and population impacts remains poorly described. Here we show that the extent of the environmental reservoir explains broad patterns of host infection and the severity of disease impacts of a virulent pathogen. We examined reservoir and host infection dynamics and the resulting impacts of Pseudogymnoascus destructans, the fungal pathogen that causes white-nose syndrome, in 39 species of bats at 101 sites across the globe. Lower levels of pathogen in the environment consistently corresponded to delayed infection of hosts, fewer and less severe infections, and reduced population impacts. In contrast, an extensive and persistent environmental reservoir led to early and widespread infections and severe population declines. These results suggest that continental differences in the persistence or decay of P. destructans in the environment altered infection patterns in bats and influenced whether host populations were stable or experienced severe declines from this disease. Quantifying the impact of the environmental reservoir on disease dynamics can provide specific targets for reducing pathogen levels in the environment to prevent or control future epidemics.

Comparing context-dependent call sequences employing machine learning methods: an indication of syntactic structure of greater horseshoe bats.

For analysis of vocal syntax, accurate classification of call sequence structures in different behavioural contexts is essential. However, an effective, intelligent program for classifying call sequences from numerous recorded sound files is still lacking. Here, we employed three machine learning algorithms (logistic regression, support vector machine and decision trees) to classify call sequences of social vocalizations of greater horseshoe bats (Rhinolophus ferrumequinum) in aggressive and distress contexts. The three machine learning algorithms obtained highly accurate classification rates (logistic regression 98%, support vector machine 97% and decision trees 96%). The algorithms also extracted three of the most important features for the classification: the transition between two adjacent syllables, the probability of occurrences of syllables in each position of a sequence, and the characteristics of a sequence. The results of statistical analysis also supported the classification of the algorithms. The study provides the first efficient method for data mining of call sequences and the possibility of linguistic parameters in animal communication. It suggests the presence of song-like syntax in the social vocalizations emitted within a non-breeding context in a bat species.

Seasonal Changes in Gut Microbiota Diversity and Composition in the Greater Horseshoe Bat.

A large number of microorganisms colonize the intestines of animals. The gut microbiota plays an important role in nutrient metabolism and affects a number of physiological mechanisms in the host. Studies have shown that seasonal changes occur in the intestinal microbes of mammals that hibernate seasonally. However, these studies only focused on ground squirrels and bears. It remains unclear how hibernation might affect the intestinal microbes of bats. In this study, we measured microbial diversity and composition in the gut of Rhinolophus ferrumequinum in different periods (early spring, early summer, late summer, torpor, and interbout arousal) using 16S ribosomal RNA gene amplicon sequencing and PICRUSt to predict functional profiles. We found seasonal changes in the diversity and composition of the gut microbes in R. ferrumequinum. The diversity of gut microbiota was highest in the late summer and lowest in the early summer. The relative abundance of Proteobacteria was highest in the early summer and significantly lower in other periods. The relative abundance of Firmicutes was lowest in the early summer and significantly increased in the late summer, followed by a significant decrease in the early winter and early spring. The relative abundance of Tenericutes was significantly higher in the early spring compared with other periods. The results of functional prediction by PICRUSt showed seasonal variations in the relative abundance of metabolism-related pathways, including lipid metabolism, carbohydrate metabolism, and energy metabolism. Functional categories for carbohydrate metabolism had significantly lower relative abundance in early winter-torpor compared with late summer, while those associated with lipid metabolism had significantly higher relative abundance in the early winter compared with late summer. Overall, our results show that seasonal physiological changes associated with hibernation alter the gut microbial community of R. ferrumequinum. Hibernation may also alter the metabolic function of intestinal microbes, possibly by converting the gut microflora from carbohydrate-related to lipid-related functional categories. This study deepens our understanding of the symbiosis between hibernating mammals and gut microbes.

The roles of morphological traits, resource variation and resource partitioning associated with the dietary niche expansion in the fish-eating bat Myotis pilosus.

Niche expansion and shifts are involved in the response and adaptation to environmental changes. However, it is unclear how niche breadth evolves and changes toward higher-quality resources. Myotis pilosus is both an insectivore and a piscivore. We examined the dietary composition and seasonality in M. pilosus and the closely related Myotis fimbriatus using next-generation DNA sequencing. We tested whether resource variation or resource partitioning help explain the dietary expansion from insects to fish in M. pilosus. While diet composition and diversity varied significantly between summer and autumn, the proportion of fish-eating individuals did not significantly change between seasons in M. pilosus. Dietary overlap between M. pilosus and M. fimbriatus during the same seasons was much higher than within individual species across seasons. We recorded a larger body size, hind foot length, and body mass in M. pilosus than in M. fimbriatus and other insectivorous trawling bats from China. Similar morphological differences were found between worldwide fishing bats and nonfishing trawling bats. Our results suggest that variation in insect availability or interspecific competition may not play important roles in the dietary expansion from insects to fish in M. pilosus. Myotis pilosus has morphological advantages that may help it use fish as a diet component. The morphological advantage promoting dietary niche evolution toward higher quality resources may be more important than variation in the original resource and the effects of interspecific competition.