Recombination analysis on the receptor switching event of MERS-CoV and its close relatives: implications for the emergence of MERS-CoV.

BACKGROUND: PlMERS-CoV is a coronavirus known to cause severe disease in humans, taxonomically classified under the subgenus Merbecovirus. Recent findings showed that the close relatives of MERS-CoV infecting vespertillionid bats (family Vespertillionidae), named NeoCoV and PDF-2180, use their hosts' ACE2 as their entry receptor, unlike the DPP4 receptor usage of MERS-CoV. Previous research suggests that this difference in receptor usage between these related viruses is a result of recombination. However, the precise location of the recombination breakpoints and the details of the recombination event leading to the change of receptor usage remain unclear. METHODS: We used maximum likelihood-based phylogenetics and genetic similarity comparisons to characterise the evolutionary history of all complete Merbecovirus genome sequences. Recombination events were detected by multiple computational methods implemented in the recombination detection program. To verify the influence of recombination, we inferred the phylogenetic relation of the merbecovirus genomes excluding recombinant segments and that of the viruses' receptor binding domains and examined the level of congruency between the phylogenies. Finally, the geographic distribution of the genomes was inspected to identify the possible location where the recombination event occurred. RESULTS: Similarity plot analysis and the recombination-partitioned phylogenetic inference showed that MERS-CoV is highly similar to NeoCoV (and PDF-2180) across its whole genome except for the spike-encoding region. This is confirmed to be due to recombination by confidently detecting a recombination event between the proximal ancestor of MERS-CoV and a currently unsampled merbecovirus clade. Notably, the upstream recombination breakpoint was detected in the N-terminal domain and the downstream breakpoint at the S2 subunit of spike, indicating that the acquired recombined fragment includes the receptor-binding domain. A tanglegram comparison further confirmed that the receptor binding domain-encoding region of MERS-CoV was acquired via recombination. Geographic mapping analysis on sampling sites suggests the possibility that the recombination event occurred in Africa. CONCLUSION: Together, our results suggest that recombination can lead to receptor switching of merbecoviruses during circulation in bats. These results are useful for future epidemiological assessments and surveillance to understand the spillover risk of bat coronaviruses to the human population.

Substantial viral diversity in bats and rodents from East Africa: insights into evolution, recombination, and cocirculation.

BACKGROUND: Zoonotic viruses cause substantial public health and socioeconomic problems worldwide. Understanding how viruses evolve and spread within and among wildlife species is a critical step when aiming for proactive identification of viral threats to prevent future pandemics. Despite the many proposed factors influencing viral diversity, the genomic diversity and structure of viral communities in East Africa are largely unknown. RESULTS: Using 38.3 Tb of metatranscriptomic data obtained via ultradeep sequencing, we screened vertebrate-associated viromes from 844 bats and 250 rodents from Kenya and Uganda collected from the wild. The 251 vertebrate-associated viral genomes of bats (212) and rodents (39) revealed the vast diversity, host-related variability, and high geographic specificity of viruses in East Africa. Among the surveyed viral families, Coronaviridae and Circoviridae showed low host specificity, high conservation of replication-associated proteins, high divergence among viral entry proteins, and frequent recombination. Despite major dispersal limitations, recurrent mutations, cocirculation, and occasional gene flow contribute to the high local diversity of viral genomes. CONCLUSIONS: The present study not only shows the landscape of bat and rodent viromes in this zoonotic hotspot but also reveals genomic signatures driven by the evolution and dispersal of the viral community, laying solid groundwork for future proactive surveillance of emerging zoonotic pathogens in wildlife. Video Abstract.

Cross-species spill-over potential of the H9N2 bat influenza A virus.

In 2017, a novel influenza A virus (IAV) was isolated from an Egyptian fruit bat. In contrast to other bat influenza viruses, the virus was related to avian A(H9N2) viruses and was probably the result of a bird-to-bat transmission event. To determine the cross-species spill-over potential, we biologically characterize features of A/bat/Egypt/381OP/2017(H9N2). The virus has a pH inactivation profile and neuraminidase activity similar to those of human-adapted IAVs. Despite the virus having an avian virus-like preference for α2,3 sialic acid receptors, it is unable to replicate in male mallard ducks; however, it readily infects ex-vivo human respiratory cell cultures and replicates in the lungs of female mice. A/bat/Egypt/381OP/2017 replicates in the upper respiratory tract of experimentally-infected male ferrets featuring direct-contact and airborne transmission. These data suggest that the bat A(H9N2) virus has features associated with increased risk to humans without a shift to a preference for α2,6 sialic acid receptors.

Bat-borne H9N2 influenza virus evades MxA restriction and exhibits efficient replication and transmission in ferrets.

Influenza A viruses (IAVs) of subtype H9N2 have reached an endemic stage in poultry farms in the Middle East and Asia. As a result, human infections with avian H9N2 viruses have been increasingly reported. In 2017, an H9N2 virus was isolated for the first time from Egyptian fruit bats (Rousettus aegyptiacus). Phylogenetic analyses revealed that bat H9N2 is descended from a common ancestor dating back centuries ago. However, the H9 and N2 sequences appear to be genetically similar to current avian IAVs, suggesting recent reassortment events. These observations raise the question of the zoonotic potential of the mammal-adapted bat H9N2. Here, we investigate the infection and transmission potential of bat H9N2 in vitro and in vivo, the ability to overcome the antiviral activity of the human MxA protein, and the presence of N2-specific cross-reactive antibodies in human sera. We show that bat H9N2 has high replication and transmission potential in ferrets, efficiently infects human lung explant cultures, and is able to evade antiviral inhibition by MxA in transgenic B6 mice. Together with its low antigenic similarity to the N2 of seasonal human strains, bat H9N2 fulfils key criteria for pre-pandemic IAVs.

Ecology shapes the genomic and biosynthetic diversification of Streptomyces bacteria from insectivorous bats.

Streptomyces are prolific producers of secondary metabolites from which many clinically useful compounds have been derived. They inhabit diverse habitats but have rarely been reported in vertebrates. Here, we aim to determine to what extent the ecological source (bat host species and cave sites) influence the genomic and biosynthetic diversity of Streptomyces bacteria. We analysed draft genomes of 132 Streptomyces isolates sampled from 11 species of insectivorous bats from six cave sites in Arizona and New Mexico, USA. We delineated 55 species based on the genome-wide average nucleotide identity and core genome phylogenetic tree. Streptomyces isolates that colonize the same bat species or inhabit the same site exhibit greater overall genomic similarity than they do with Streptomyces from other bat species or sites. However, when considering biosynthetic gene clusters (BGCs) alone, BGC distribution is not structured by the ecological or geographical source of the Streptomyces that carry them. Each genome carried between 19-65 BGCs (median=42.5) and varied even among members of the same Streptomyces species. Nine major classes of BGCs were detected in ten of the 11 bat species and in all sites: terpene, non-ribosomal peptide synthetase, polyketide synthase, siderophore, RiPP-like, butyrolactone, lanthipeptide, ectoine, melanin. Finally, Streptomyces genomes carry multiple hybrid BGCs consisting of signature domains from two to seven distinct BGC classes. Taken together, our results bring critical insights to understanding Streptomyces-bat ecology and BGC diversity that may contribute to bat health and in augmenting current efforts in natural product discovery, especially from underexplored or overlooked environments.

Bat species assemblage predicts coronavirus prevalence.

Anthropogenic disturbances and the subsequent loss of biodiversity are altering species abundances and communities. Since species vary in their pathogen competence, spatio-temporal changes in host assemblages may lead to changes in disease dynamics. We explore how longitudinal changes in bat species assemblages affect the disease dynamics of coronaviruses (CoVs) in more than 2300 cave-dwelling bats captured over two years from five caves in Ghana. This reveals uneven CoV infection patterns between closely related species, with the alpha-CoV 229E-like and SARS-related beta-CoV 2b emerging as multi-host pathogens. Prevalence and infection likelihood for both phylogenetically distinct CoVs is influenced by the abundance of competent species and naïve subadults. Broadly, bat species vary in CoV competence, and highly competent species are more common in less diverse communities, leading to increased CoV prevalence in less diverse bat assemblages. In line with the One Health framework, our work supports the notion that biodiversity conservation may be the most proactive measure to prevent the spread of pathogens with zoonotic potential.

Detection of ectoparasites and investigation of infection by Rickettsia spp. and Trypanosoma spp. in bats from Rio Grande do Sul, Brazil.

This study aimed to investigate the presence of ectoparasites and the occurrence of natural infection by Rickettsia spp. and Trypanosoma spp. in bats from Rio Grande do Sul (RS), Brazil. The evaluated animals were obtained from the Instituto de Pesquisas Veterinárias Desidério Finamor, sent by the Centro Estadual de Vigilância Sanitária, to carry out rabies diagnostic tests, during the period from 2016 to 2021. The bats came from 34 municipalities in RS. Of the 109 animals surveyed, 35.8% (39/109) had 385 ectoparasites, with an average of 9.9 parasites per animal. Of these bats, all had insectivorous feeding habits, with 35.9% (14/39) females and 64.1% (25/39) males. The co-parasitism of Chirnyssoides sp., Ewingana inaequalis, and Chiroptonyssus robustipes on Molossus currentium (Mammalia, Chiroptera) was recorded for the first time. All bats surveyed were negative for infection by the protozoan and bacteria. Thus, the expansion of the occurrence of these ectoparasites in insectivorous bats in RS was observed. Furthermore, this study corresponds to the first recorded interspecific associations for the species.

Assessing the emergence time of SARS-CoV-2 zoonotic spillover.

Understanding the evolution of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) and its relationship to other coronaviruses in the wild is crucial for preventing future virus outbreaks. While the origin of the SARS-CoV-2 pandemic remains uncertain, mounting evidence suggests the direct involvement of the bat and pangolin coronaviruses in the evolution of the SARS-CoV-2 genome. To unravel the early days of a probable zoonotic spillover event, we analyzed genomic data from various coronavirus strains from both human and wild hosts. Bayesian phylogenetic analysis was performed using multiple datasets, using strict and relaxed clock evolutionary models to estimate the occurrence times of key speciation, gene transfer, and recombination events affecting the evolution of SARS-CoV-2 and its closest relatives. We found strong evidence supporting the presence of temporal structure in datasets containing SARS-CoV-2 variants, enabling us to estimate the time of SARS-CoV-2 zoonotic spillover between August and early October 2019. In contrast, datasets without SARS-CoV-2 variants provided mixed results in terms of temporal structure. However, they allowed us to establish that the presence of a statistically robust clade in the phylogenies of gene S and its receptor-binding (RBD) domain, including two bat (BANAL) and two Guangdong pangolin coronaviruses (CoVs), is due to the horizontal gene transfer of this gene from the bat CoV to the pangolin CoV that occurred in the middle of 2018. Importantly, this clade is closely located to SARS-CoV-2 in both phylogenies. This phylogenetic proximity had been explained by an RBD gene transfer from the Guangdong pangolin CoV to a very recent ancestor of SARS-CoV-2 in some earlier works in the field before the BANAL coronaviruses were discovered. Overall, our study provides valuable insights into the timeline and evolutionary dynamics of the SARS-CoV-2 pandemic.

Genomic characterization of a novel Hepatovirus identified in Maranhão state, Brazil.

Bats are efficient reservoirs of a number of viruses with zoonotic potential, and are involved directly in the transmission cycle of many zoonoses. In the present study, which is part of a larger project that is documenting the viromes of the bat species found in the Mid-North states of Maranhão and Piauí, we analyzed 16 pooled samples obtained from four species of bat of the genus Artibeus-Artibeus obscurus, Artibeus cinereus, Artibeus lituratus and Artibeus planirostris. We describe and identify a Hepatovirus, denominated Hepatovirus H isolate sotense, which was found in a pool of internal organs (liver and lungs) extracted from a specimen of A. planirostris, a frugivorous bat, collected in the Cerrado biome of Maranhão state. This material was analyzed using new generation sequencing, which produced a contig of 7390 nucleotides and presented a degree of identity with a number of existing Hepatovirus sequences available for bats (amino acid identity of 61.5% with Bat hepatovirus C of Miniopterus cf. manavi, 66.6% with Bat hepatovirus G of Coleura afra, 67.4% with Hepatovirus G2 of Rhinolophus landeri, and 75.3% with Hepatovirus H2 of Rhinolophus landeri). The analysis of the functional domains of this contig confirmed a pattern consistent with the characteristics of the genus Hepatovirus (Picornaviridae). In the phylogenetic tree with several other Hepatovirus species, this genome also grouped in a monophyletic clade with Hepatovirus H (HepV-H1; HepV-H2, and HepV-H3) albeit on an external branch, which suggests that it may be a distinct genotype within this species. This is the first isolate of Hepatovirus H identified in bats from South America, and represents an important discovery, given that most studies of viruses associated with bats in the state of Maranhão have focused on the family Rhabdoviridae.

Correlated evolution between body size and echolocation in bats (order Chiroptera).

BACKGROUND: Body size and echolocation call frequencies are related in bats. However, it is unclear if this allometry applies to the entire clade. Differences have been suggested between nasal and oral emitting bats, as well as between some taxonomic families. Additionally, the scaling of other echolocation parameters, such as bandwidth and call duration, needs further testing. Moreover, it would be also interesting to test whether changes in body size have been coupled with changes in these echolocation parameters throughout bat evolution. Here, we test the scaling of peak frequency, bandwidth, and call duration with body mass using phylogenetically informed analyses for 314 bat species. We specifically tested whether all these scaling patterns differ between nasal and oral emitting bats. Then, we applied recently developed Bayesian statistical techniques based on large-scale simulations to test for the existence of correlated evolution between body mass and echolocation. RESULTS: Our results showed that echolocation peak frequencies, bandwidth, and duration follow significant allometric patterns in both nasal and oral emitting bats. Changes in these traits seem to have been coupled across the laryngeal echolocation bats diversification. Scaling and correlated evolution analyses revealed that body mass is more related to peak frequency and call duration than to bandwidth. We exposed two non-exclusive kinds of mechanisms to explain the link between size and each of the echolocation parameters. CONCLUSIONS: The incorporation of Bayesian statistics based on large-scale simulations could be helpful for answering macroevolutionary patterns related to the coevolution of traits in bats and other taxonomic groups.

A comparative study on eDNA-based detection of Siamese bat catfish (Oreoglanis siamensis) in wet and dry conditions.

The use of environmental DNA (eDNA) analysis has demonstrated notable efficacy in detecting the existence of freshwater species, including those that are endangered or uncommon. This application holds significant potential for enhancing environmental monitoring and management efforts. However, the efficacy of eDNA-based detection relies on several factors. In this study, we assessed the impact of rainfall on the detection of eDNA for the Siamese bat catfish (Oreoglanis siamensis). Quantitative polymerase chain reaction (qPCR) analysis indicated that samples from days with average rainfall exceeding 35 mm (classified as heavy and very heavy rain) yielded negative results. While eDNA detection remains feasible on light or moderate rainy days, a noteworthy reduction in eDNA concentration and qPCR-positive likelihood was observed. Analysis across 12 sampling sites established a statistically significant negative relationship (p < 0.001) between eDNA detection and rainfall. Specifically, for each 1 mm increase in rainfall, there was an observed drop in eDNA concentration of 0.19 copies/mL (±0.14). The findings of this study provide definitive evidence that precipitation has a significant impact on the detection of eDNA in Siamese bat catfish. However, in the case of adverse weather conditions occurring on the day of sampling, our research indicates that it is acceptable to continue with the task, as long as the rainfall is not heavy or very heavy. To enhance the effectiveness of an eDNA survey, it is crucial to consider many factors related to climatic conditions. The aforementioned factor holds significant importance not only for the specific species under scrutiny but also for the broader dynamics of the climate.

Hierarchically embedded scales of movement shape the social networks of vampire bats.

Social structure can emerge from hierarchically embedded scales of movement, where movement at one scale is constrained within a larger scale (e.g. among branches, trees, forests). In most studies of animal social networks, some scales of movement are not observed, and the relative importance of the observed scales of movement is unclear. Here, we asked: how does individual variation in movement, at multiple nested spatial scales, influence each individual's social connectedness? Using existing data from common vampire bats (Desmodus rotundus), we created an agent-based model of how three nested scales of movement-among roosts, clusters and grooming partners-each influence a bat's grooming network centrality. In each of 10 simulations, virtual bats lacking social and spatial preferences moved at each scale at empirically derived rates that were either fixed or individually variable and either independent or correlated across scales. We found that numbers of partners groomed per bat were driven more by within-roost movements than by roost switching, highlighting that co-roosting networks do not fully capture bat social structure. Simulations revealed how individual variation in movement at nested spatial scales can cause false discovery and misidentification of preferred social relationships. Our model provides several insights into how nonsocial factors shape social networks.

The effect of urbanization on species composition and trophic guilds of bats (Mammalia, Chiroptera) in the Brazilian Savanna.

Urban environments present less environmental heterogeneity in relation to the natural ones, affecting the biodiversity of bats and the ecological processes in which they participate. In this way, we will identify how urbanization influences the structure of bat communities in the municipality of Goiânia, Goiás, Brazil. We compared species composition, guilds and bat richness in a gradient that crossed urban, semi-urban and natural areas in the municipality of Goiânia, contained in the Cerrado biome. We captured a total of 775 bats of 16 species distributed in three families. Urban areas had a higher species abundance, while semi-urban areas had a higher species richness. The three types of environments have different compositions, the urban one being more homogeneous, the fauna in these areas is composed of generalist species, which benefit from this process. The diversity present in semi-urban areas is a consequence of the intersection between urban and natural fauna, which is why urban expansion needs to occur in a planned manner to minimize the impacts of this process and ensure the maintenance of biodiversity.

Biomonitoring of benzophenones in guano samples of wild bats in Poland.

Benzophenones (BPs) are substances used in the production of sunscreens, cosmetics, and personal care products. However, there is a lack of knowledge of BPs in wild animals. Therefore, the study aimed to assess the concentration of selected BPs commonly used in the cosmetic industry in guano samples collected from 4 colonies of greater mouse-eared bats (Myotis myotis). Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to determine guano concentrations of benzophenone 1 (BP-1), benzophenone 2 (BP-2), benzophenone 3 (BP-3) and benzophenone 8 (BP-8). BP-1 levels above the method quantification limit (MQL) were noted in 97.5% of samples and fluctuated from <0.1 ng/g to 259 ng/g (mean 41.50 ng/g, median 34.8). The second most common was BP-3, which fluctuated from <0.1 ng/g to 19 ng/g (mean 6.67 ng/g, median 5.05), and its levels higher than MQL were observed in 40% of samples. BP-2 and BP-8 concentrations did not exceed the method detection limit (0.04 ng/g) in any analyzed sample. There were visible differences in the BP-1 and BP-3 levels among the studied bat colonies. Mean BP-1 concentration fluctuated from 11.23±13.13 ng/g to 76.71±65.51 ng/g and differed significantly between the colonies. Mean BP-3 concentration fluctuated from 5.03±6.03 ng/g to 9.18±7.65 mg/g, but it did not differ significantly between the colonies. The results show that guano is a suitable matrix for the assessment of wildlife exposure to BPs. This could be particularly advantageous in protected species, where not disturbing and stressing the animals are crucial.

DNA Damage in Bat Blood Leukocytes Using a Chromatin Dispersion Test (CDT): Biomarker of Environmental Genotoxicity.

Environmental pollutants produce adverse effects on organisms and ecosystems. Biomonitoring and biomarkers offer a reasonable approach to make these assessments. Induced genetic changes can be using as a biomarker in organisms that react to a given compound in the ecosystem. Monitoring environmental genotoxicity necessitates the choice of model animals known as "sentinels or biological monitors" and the suitability of validated tests for DNA damage evaluation. We aimed to estimate the DNA damage produced by thermal stress in the leukocytes of the Mexican free-tailed bat (Tadarida brasiliensis). The DNA damage in bat leukocytes exposed to different temperatures (35 °C, 45 °C, and 55 °C) was evaluated by the adapted chromatin dispersion test (CDT) and the results were confirmed by the alkaline comet test. The CDT permitted a clear representation of leukocytes with fragmented DNA and of nonfragmented DNA. In addition, we detected nuclear anomalies in relation to cell death cellular swelling, nuclear fragmentation, and chromatin lysis. The alkaline comet assay revealed that the halos of diffuse chromatin include fragmented DNA. The assay of the method employing the CDT is well established, precise, and cost-effective for the routine quantitative analysis of DNA damage on the effect of the leukocytes of bats exposed to thermal stress. This could also apply as a sensitive screening tool for the evaluation of genotoxicity in environmental protection programs.

Deforestation and Bovine Rabies Outbreaks in Costa Rica, 1985-2020.

In Latin America, rabies virus has persisted in a cycle between Desmodus rotundus vampire bats and cattle, potentially enhanced by deforestation. We modeled bovine rabies virus outbreaks in Costa Rica relative to land-use indicators and found spatial-temporal relationships among rabies virus outbreaks with deforestation as a predictor.

Conserved Functions of Orthohepadnavirus X Proteins to Inhibit Type-I Interferon Signaling.

Orthohepadnavirus causes chronic hepatitis in a broad range of mammals, including primates, cats, woodchucks, and bats. Hepatitis B virus (HBV) X protein inhibits type-I interferon (IFN) signaling, thereby promoting HBV escape from the human innate immune system and establishing persistent infection. However, whether X proteins of Orthohepadnavirus viruses in other species display a similar inhibitory activity remains unknown. Here, we investigated the anti-IFN activity of 17 Orthohepadnavirus X proteins derived from various hosts. We observed conserved activity of Orthohepadnavirus X proteins in inhibiting TIR-domain-containing adaptor protein inducing IFN-ß (TRIF)-mediated IFN-ß signaling pathway through TRIF degradation. X proteins from domestic cat hepadnavirus (DCH), a novel member of Orthohepadnavirus, inhibited mitochondrial antiviral signaling protein (MAVS)-mediated IFNß signaling pathway comparable with HBV X. These results indicate that inhibition of IFN signaling is conserved in Orthohepadnavirus X proteins.

Morphological and molecular characteristics of Paralecithodendrium longiforme (Digenea: Lecithodendriidae) adults and cercariae from Chinese pipistrelle bats and viviparid snails in Thailand.

This study aimed to describe the morphological and molecular characteristics of Paralecithodendrium longiforme (Digenea: Lecithodendriidae) adults and cercariae isolated in Thailand. Adult flukes were isolated from the Chinese pipistrelle bat (Hypsugo sp.), and cercariae were detected in the viviparid snail (Filopaludina martensi martensi) from Chiang Mai province. The morphological characteristics were observed and described using conventional methods, and the molecular characteristics with internal transcribed spacer 2 (ITS2) and 28S rDNA gene sequences. The adult flukes were fusiform, 0.84-0.98 mm in length, and 0.37-0.49 mm in width, and were distinguishable from other species by the presence of longitudinal uterine coils. The cercariae were nonvirgulate xiphidiocercariae, with the oral sucker bigger than the acetabulum, the tail without fin fold, a body size of 117.5-138.3 × 48.3-52.2 µm, and a tail size of 100.7-103.7 × 15.0-18.9 µm. Molecular studies revealed that the adults and cercariae shared 99.3% (ITS2) and 99.6% (28S rDNA) homology with each other. They were phylogenetically close to P. longiforme with an identity of 94.5% for ITS2 and 98.7% for 28S rDNA. This study provides new information on the natural definitive host and first intermediate host of P. longiforme in Thailand. The discovery of its cercarial stage in Filopaludina snails highlights the importance of monitoring the associated second intermediate host and prevention and control of this potentially zoonotic trematode.

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.