Gut microbial composition changes in an Astrovirus-infected Neotropical frugivorous bat – a one health perspective

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Brändel, Dominik W. Melville, Kerstin Wilhelm, Victor M. Corman, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8419449/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 13 You are reading this latest preprint version Abstract Astroviruses are becoming a growing concern in public and veterinary health. In humans, astrovirus infections can cause severe diarrhea and may lead to neuropathological encephalitis, whereas in wildlife, these enteropathogenic viral infections often lack overt symptoms and, thus, remain unnoticed. Yet, their close interaction with the host’s gastrointestinal microbiome might drive cascading effects with disadvantages for host health. Bats harbor many zoonotic viruses without showing signs of disease, and many species move freely along the gradient from pristine to agricultural landscapes. To better understand the impact of Astrovirus (AstV) infection under a One Health framework, we investigated the gut microbiome of naturally AstV-infected Seba’s short-tailed bats ( Carollia perspicillata ; n = 234) inhabiting old-growth lowland forests or forest fragments embedded in an agricultural matrix in Panama. AstV prevalence was higher in forest fragments. We observed that AstV infection is associated with a shift in microbial beta but not alpha diversity, which points towards the replacement of common gut microbial taxa when infected. Indeed, potential beneficial bacteria, such as Lactococcus , decreased in abundance, whereas potentially pathogenic bacteria from the Helicobacter genus increased in AstV-positive bats. Two Helicobacter haplotypes closely related to avian Helicobacter species were identified. We conclude that even though the impact of infection on the microbiome was not amplified in forest fragments, the higher infection likelihood in landscapes altered by humans implies more frequent or prolonged health repercussions for bats. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Astroviruses (AstVs) are positive-sense, single-stranded enteric RNA viruses with an incredibly broad host range across diverse avian and mammalian hosts, including humans, and are directly and easily transmitted via the fecal-oral route [ 1 , 2 ]. The recent discovery of numerous novel astrovirus strains, their large genetic diversity and ability for cross-species recombination, highlight the risk of interspecies or even zoonotic transmission of AstVs [ 3 – 6 ]. In wildlife, AstV infections often persist asymptomatically, whereas in humans, infections can cause severe diarrhea and may lead to neuropathological encephalitis [ 7 ]. Symptoms of AstV infection vary depending on age and immunological status of infected hosts with children and immunosuppressed being most affected [ 1 ]. Land-use changes and, as a consequence, increased likelihood of human-wildlife contacts amplify the chances of disease spread beyond species boundaries [ 8 , 9 ]. Especially, food and nutrient scarcity in man-made landscapes increases overlap between wildlife, domesticated animals and humans [ 10 – 12 ]. Prominent examples include the regular spillover events of the zoonotic Nipah and Hendra viruses from fruit bats to pigs and horses [ 11 , 13 ], and the repeated transmission of morbilli or lyssaviruses from vampire bats to pigs and cattle [ 14 , 15 ]. The high degree of population fragmentation in mosaic landscapes and the frequent crossover with novel hosts may even accelerate virus evolution, exacerbating their zoonotic risk [ 16 , 17 ]. Besides, the health consequences for wildlife inhabiting fragmented forests, agricultural landscapes or urban environments are often poorly understood or entirely unknown [ 18 – 21 ]. The negative health effects from enteropathogenic viral infections, such as those caused by AstVs, may arise from cascading effects on the host’s gastrointestinal microbiome, enhancing viral activity or facilitating co-infections (e.g., norovirus [ 22 ], rotavirus [ 23 ], coronavirus [ 24 ], adenovirus [ 25 ], and astrovirus [ 26 – 29 ]). The gastrointestinal microbiome is an incredibly complex and highly diverse ecosystem, with important roles in nutrient acquisition (e.g., vitamins; [ 30 ]), toxin degradation [ 31 ] and metabolism [ 32 ] as well as in the defense against enteropathogens [ 33 ], and the development and maintenance of immune responses [ 34 – 36 ]. Abnormal shifts in its diversity and composition, beyond the normal range of intraspecific variation [ 37 ], could be either cause or consequence of the susceptibility to pathogens or chronic inflammatory diseases [ 38 – 40 ]. For instance, increased viral load and co-infections amplify gut microbial dysbiosis in bats and voles [ 41 , 42 ]. However, the interaction between viruses and the hosts’ microbiome is an essential, yet often overlooked, component in the holobiont machinery [ 43 – 45 ]. Astroviruses are the fourth most common virus family recovered from bats, and a high diversity of AstVs have been reported in bat species worldwide [ 46 – 49 ]. Due to their high prevalence and presumably mild pathogenicity AstVs represent a perfect study system to understand potential health effects in visibly symptomless bats. At the same time, bats are seen as a model system to investigate the role of microbes in host evolution, physiology and fitness as they harbor a diverse set of microbiomes likely reflecting their ecological and phylogenetic diversity ([ 50 ] but see [ 51 ] and discussion in [ 52 ]). Unsurprising therefore, that a previous study identified AstV-mediated disruption of the gut microbial community in the Jamaican fruit bat ( Artibeus jamaicensis ) [ 28 ]. Little is, however, known about the health consequences of land-use changes for bats [ 53 – 55 ], and the role of disease ecology in that context. From a OneHealth perspective, a better understanding of how land-use change, host microbial health and infection ecology interact is pivotal to predict and prevent spillover events, and safeguard human and animal wellbeing [ 56 ]. In this study, we investigated the effect and interplay of AstV infection and land-use change on the gut microbiome diversity and composition in a neotropical frugivorous habitat generalist, the Seba´s short-tailed bat ( Carollia perspicillata ). The species is abundant throughout Central and South America, often profiting from disturbed areas and the abundance of fruits of early succession, hence found in human-modified habitats, even often roosting close to rural settlements [ 57 , 58 ]. As the microbiome is the first line of defense in the gut and linked to physiological stress responses, we hypothesized that AstV-infections and human disturbance reduce microbial alpha diversity, shift microbial beta diversity, and favor the emergence of (opportunistically) pathogenic bacteria. Age may also play a role, as shown in A. jamaicensis [ 28 ], immunologically naïve subadults exhibited altered gut microbiota when infected with AstVs. Such age-dependent responses may therefore be common in bats. Moreover, we predict that the effect on the microbiota is amplified in AstV-positive bats inhabiting forest fragments embedded in an agricultural matrix. Methods Focus species and fecal sample collection The phyllostomid bat C. perspicillata is a widespread frugivore (Fig. 1 A), found in high abundance across large parts of Central and South America [ 59 ]. C. perspicillata consumes fruits of early successional plants, e.g., pepper plants (genus: Piper ) [ 60 , 61 ]. The species shows flexible roosting behavior in crevices and hollows, while flight capabilities limit the species to searching for food in relative proximity to its roost (mean: 1.6 km) [ 57 , 62 , 63 ]. Its diet preference for fruits from successional plants found frequently in agricultural landscapes cleared for livestock does not only allows the species to persist but even to thrive in human-disturbed habitats [ 57 ]. Bats were caught in lowland tropical rainforest ecosystems of Central Panama using mist-nets (ECOTONE, Gdynia, Poland) in a standardized sampling design at five independent sampling sites in three landscapes varying in degree of fragmentation and human disturbance: natural lowland forest, forest fragments embedded in an agricultural matrix, and forested islands surrounded by water [ 57 ]. However, C. perspicillata is rarely found on islands [ 57 , 64 , 65 ]. Thus, the work here only concerns sites in old-growth forest and forest fragments surrounded by agricultural landscapes. After capture, individuals were sexed, and age was determined based on the ossification of the epiphyses of the digits [ 66 ]. Individual fecal samples were collected directly from bats upon defecation during handling or collected out of the fabric bags, in which bats were kept temporarily before processing, and immediately preserved in RNAlater (Life Technologies). All bats were released on site right after handling. Capture and sampling techniques followed Panamanian protocols (MiAmbiente, Republica de Panama: SE/A-75-13 to SE/A-28-17) and were ethically certified by the Smithsonian Tropical Research Institute (IACUC protocols: 2014-0101-2016 to 2016-0627-2019). Astrovirus testing Viral RNA was extracted from 434 fecal samples using the MagNA Pure 96 DNA and the Viral NA Small Volume Kit (Roche) corresponding to the manufacturer’s guidelines. AstV prevalence was determined by the broadly reactive nested reverse transcription-PCR (RT-PCR) assay, as described previously [ 28 ]. Bacterial DNA extraction and 16S rRNA gene bacterial amplicon sequencing Microbiome analyses were carried out in a total of 234 individuals. After homogenization of fecal samples (SpeedMill PLUS Homogenizer, Analytik Jena, Germany), bacterial genomic DNA was extracted applying the NucleoSpin 96 Soil kit (Macherey-Nagel, Germany) following the manufacturer's instructions. The universal 16S primers 515F/806R were used for PCR amplification of the hypervariable V4 region (291 bp) of 16S rRNA – a standard primer pair, which leads to very high bacterial diversity and performed best in a recent primer comparison experiment [ 67 ]. We used the Fluidigm System (Access Array™ System for Illumina Sequencing Systems, ©Fluidigm Corporation) for primer tagging. PCRs (15 µl reaction volume) were executed as described previously [ 28 , 68 ]. After purification (NucleoMag bead-based size selection, Macherey-Nagel, Germany) and quantification of barcoded samples (DropSense, Trinean, US), we applied paired-end sequencing on the Illumina® MiSeq platform. Bioinformatics All subsequent analyses were performed within R (v4.4.1, [ 69 ]). We applied the DADA2 pipeline [ 70 ]. Reads were trimmed from both ends to remove low-quality regions, and after denoising and merging, chimeric sequences were removed, following the ‘consensus’ method as implemented in DADA2 . A naïve Bayesian classifier within SILVA v138 database was applied for taxonomic assignments of obtained nonchimeric amplicon sequence variants (ASVs). The phyloseq package (v1.48, [ 71 ]) was further used for data processing. ASVs assigned to chloroplasts, mitochondria, and unassigned ASVs at the phylum level were removed from the dataset, and cleaned using the MicroViz package (v0.12.5; [ 72 ]). Astrovirus prevalence First, we calculated the average prevalence of AstV among 434 virus-screened C. perspicillata for each sampling site. We compared mean prevalence (square-root transformed to meet normality assumption) using one-sided t-test with landscape (continuous forests vs. forested fragments in an agricultural matrix) as explanatory variable. Gut microbial composition and alpha-diversity We calculated the gut microbial alpha diversity indices, the number of observed ASVs, Shannon index and Faith’s phylogenetic diversity, after rarefying the data to 5000 sequences per sample. Rarefying removed 35 samples with fewer than 5000 reads (final sample size: n = 199; [ 73 ]), although unrarefied data yielded comparable results (Supplementary Fig. 1). To analyze the effects on alpha diversity metrics (Faith’s PD and Observed ASVs were log-transformed to meet normality assumptions), we used linear mixed-effects models (LMERs) using the lme4 package (v1.1-37; [ 74 ]) including AstV-infection (positive vs. negative), age (subadults vs. adults), sex (male vs. female), season (wet vs. dry), and landscape (continuous forests vs. forested fragments in an agricultural matrix) as explanatory variables, while still accounting for sequencing depth and setting sampling site as random effect. Since a previous study revealed that AstV infections influenced the gut microbiome dependent on host age [ 28 ], and we aimed to unveil potential interactions between AstV-infection and landscape differences, we included both two-way interactions in each model, and back-selected. Gut microbial beta-diversity Microbial beta diversity distances (i.e., Bray-Curtis, Jaccard, Aitchison, weighted and unweighted Unifrac) were calculated for ASVs and ASVs agglomerated at genus (since higher level taxonomic assignments are often more reliable for wildlife 16S data). The gut microbial dissimilarity was compared using permutational analyses of variance (999 permutations) encoded in the vegan package (v2.7-2; [ 75 ]). We used the same explanatory variables as for the LMERs and stratified by sampling site. Principal coordinate analyses (PCoA) were performed to visualize the pattern of separation between the various categories of samples. Differential abundance analysis Finally, we performed an analysis of compositions of microbiomes with bias correction at ASV and genus levels using both AstV infection status and landscape as explanatory factors [ 76 ]. To avoid spurious results, we removed ASVs or genera prevalent in fewer than 10% of samples, as recommended [ 77 ], and applied false-discovery rate corrections [ 78 ]. Results Astrovirus prevalence and microbiome composition The prevalence of AstV infections was higher in the forest fragments surrounded by agricultural matrix (mean 22.8% ± 1.6 SE; t-value = 4.3, p = 0.002; Fig. 1 B) than in the continuous forest (mean 10.1% ± 2.5 standard error). After initial filtering, 7.188.339 sequence reads of the V4 region the bacterial 16S ribosomal RNA gene remained from 234 fecal samples of C. perspicillata . On average 17.575 (range: 1.945–110.054) high-quality reads per sample were obtained after taxonomic assignments. Rarefying to a representative number of 5.000 reads resulted in the removal of 35 samples, leaving 137 AstV-negative and 62 AstV-positive C. perspicillata for further analysis (Fig. 1 C). The dominant phylum was Pseudomonadota (mean 49.9% ± 28.9 standard deviation). At family resolution Enterobacteriaceae (mean 21.5% ± 29.4 SD) made up the highest proportion, followed by Mycoplasmataceae (mean 10.7% ± 22.1 SD) and Streptococcaceae (mean 10.1% ± 15.4 SD) of the microbial community (Fig. 2 A). Effects of AstV infection and landscape on alpha and beta diversity Neither landscape differences nor infection status significantly affected any of the alpha diversity in C. perspicillata (Supp. Table 1). There was a very weak tendency for a higher Shannon value in C. perispicillata positive for AstV in continuous forest, while lower values for AstV-positive individuals were recovered among the forest fragments (infection-landscape interaction: F 1,191 =2.83, p = 0.094; Fig. 2 B). Neither the interaction nor single effect of infection or landscape was found in any other alpha diversity metric (Supp. Table 1). Subadult C. perispicillata had a lower Shannon diversity (F 1,191 =4.52, p = 0.034). Observed ASV richness increased with sequencing depth in spite of rarefaction (Supp. Table 1). AstV infection shifted the beta diversity centroid in all distances except for weighted Unifrac (Supp. Table 2). This implies that the infection substantially affecting microbial community composition and, to a lesser extent, structure (Fig. 3 A). A weak landscape effect was only found in Aitchison distance calculated from ASV matrix (R2 = 0.010; p = 0.023), but the interaction between AstV infection and landscape was never significant. Sequencing depth affected particularly distances that weighted microbial abundances based on reads (Supp. Table 2). Beta dispersion did not differ between infection status (F 1,197 =0.14, p = 0.246; Fig. 3 B) or landscape (F 1,197 =0.07, p = 0.780; Fig. 3 C). AstV infection is associated with shifts in the relative abundance of genera and ASVs In total, 29 ASVs and 23 genera were found to be differentially abundant between AstV-positive and AstV-negative C. perspicillata (Supp. Table 3, 4): The genera Helicobacter , Leptotrichia , Neisseria and Moraxella , genera containing species pathogenic to humans or animals, were enriched in AstV-positive C. perspicillata , for instance (Fig. 4 ). Based on these results, we amplified an approximately 1200 bp long sequence in 40 randomly selected samples per landscape using a Helicobacter -specific primer pair [ 79 ], and aligned the resulting sequences to sequences from 27 Helicobacter species and Wolinella succinogenes as outgroup (see Supplementary Material for more details). Two Helicobacter haplotypes ( C.per _haplotype 1 and C.per _haplotype 2) were identified and aligned most closely to Helicobacter anseris (Fig. 4 B). By contrast, bacterial genera often beneficial to hosts, such the lactic-acid producing Lactococcus , the sugar-fermenting order of Saccharimonadales, aromatic compound degrading genus of Novosphingobium , decreased in abundance in AstV-positive bats. Although landscape effects were minimal on the whole community, certain bacteria were still recovered more abundant in continuous forests than forest fragments independent of the infection status (e.g., Paenibacillus and Actinomyces ; Supp. Table 3, 4). Discussion Bats are known to host numerous virus families with zoonotic potential [ 47 ] and harbor a particularly high diversity of astroviruses, playing a key role in AstV ecology and evolution [ 46 – 48 ]. Seba’s short-tailed bats ( C. perspicillata ) appear healthy and show no overt clinical symptoms (e.g., weakness), yet they were found to be infected with astroviruses. Astrovirus prevalence was higher in bats captured in forest fragments than in old-growth lowland rainforest sites in Panama. Furthermore, gut microbial community composition—a proxy for host health [ 80 – 82 ]—differed between AstV-negative and AstV-positive bats, suggesting that enteropathogenic viral infection influences both the taxonomic and, in all likelihood, functional diversity of the microbiome. A decline in gut microbial homeostasis during infection may increase the risk of co-infection [ 38 ], and enhance viral (and bacterial) shedding [ 42 , 83 ], posing a potential One Health concern. Consistent with this, two novel Helicobacter haplotypes closely related to H. anseris were detected in AstV-positive C. perspicillata . Human encroachment into nature is likely the main driver behind the current extinction crisis [ 84 , 85 ]. Additionally, research over the last decades has shown that land-use changes further increase the risk of disease spillover from wild animals into livestock and humans [ 8 , 9 , 16 ]. We document that AstVs were twice as likely to be detected in C. perspicillata captured in forest fragments embedded in agricultural landscapes than in individuals captured in continuous forests. This is unlike findings from the Bornean rainforest where AstV infection likelihood in insectivorous bats captured in actively logged, fragmented and recovering forest sites did not differ [ 29 ]. Similarly, landscape differences affected neither AstV prevalence [ 28 ] nor host abundance of the canopy frugivore A. jamaicensis [ 57 ]. By contrast, the understory frugivore C. perspicillata , which consumes fruits of early successional plants [ 60 , 61 ], such as the pepper plants (genus Piper ) – common in edge habitat surrounding forest fragments –, is four times more likely to be captured in forest fragments than continuous forest sites [ 57 ]. Our finding lends weight to the idea that resource availability drives spatial host densities, and that viral transmission is thus largely density mediated [ 11 , 86 , 87 ]. However, land-use change is still at the root of the rise in disease prevalence, because regionally patchy resources become more localized, amassing a higher number of hosts and, thus, increasing transmission risk. The gut microbial community of AstV-positive C. perspicillata was shifted independently of whether the bat was captured in forest fragments or old growth forest sites. This implies land-use differences do not amplify disease-mediated changes to the gut microbial community. The gut microbial community was also different in A. jamaicensis infected with AstVs although in this case the effect was dependent on host age [ 28 ]. While cause and effect are difficult to disentangle in naturally infected populations [ 38 , 43 , 80 ], AstV infections are known to alter the gut mucus barrier [ 26 ], offering a mechanism by which an enteropathogenic virus could tamper with the balance of beneficial and pathogenic gut bacteria [ 88 , 89 ]. Indeed, some beneficial bacteria, i.e., the lactic acid-producing genus Lactococcus , adhere to the outer mucus layer surrounding host epithelial cells using pili, anchoring and mucin-binding proteins [ 90 , 91 ]. A disruption of the mucus barrier may therefore have consequences for resident symbionts. While we cannot elucidate the precise mechanism, Lactococcus and other potentially beneficial gut bacteria, such as the aromatic compound degrading genus of Novosphingobium – identified to support the breakdown of defensive plant flavonoids [ 92 ] – declined in AstV-positive bats. Bacterial ASVs or genera that increased in AstV-positive bats range from commensals to pathogens, including the genera Leptotrichia and Helicobacter . Many Leptotrichia are able to ferment mono- and disaccharides to lactic acid and are found as commensal part in healthy hosts’ oral and intestinal microbiome [ 93 ]. Yet, Leptotrichia can be opportunistically pathogenic and was implicated in dental decay [ 94 ], or gut dysbiosis in hosts with bacterial or viral co-infections [ 95 , 96 ]. The bacterial genus most strongly enriched in AstV-positive bats was Helicobacter , however. Helicobacter are gram-negative bacteria able to penetrate the mucous lining of the gut due to their helical and flagellated morphology [ 97 ]. In humans, infections with Helicobacter pylori cause gastric disorders even inducing cancer [ 98 ]. Other Helicobacter species circulate in livestock and wildlife, and are often associated with gastric histopathological alterations [ 99 – 101 ]. We identified Helicobacter haplotypes clustering most closely to Helicobacter anseris , which was described in Canada geese for the first time in 2006 [ 102 ], but has since been isolated in a number of birds from South America [ 103 , 104 ]. H. anseris grows optimally in temperatures ranging between 37 and 42°C [ 102 ], which makes it feasible that H. anseris or a closely related strain could thrive in bats, which similar to birds maintain elevated body temperatures during flight [ 105 , 106 ]. In addition, bats and birds share similar gut microbial communities, which are unlike those of other mammals [ 107 ]. One could theorise that Helicobacter strains evolved in birds can infect host with a similar gut microbial community more easily. Yet, we lack histopathological information to determine if the Helicobacter haplotypes were harmful to C. perspicillata . Taken together, AstV infection in bats seem to replace beneficial bacteria with potentially pathogenic ones, undermining the services the host may derive from its microbiota. In comparison with the AstV infection, land-use differences seem to matter little to the gut microbial alpha and beta diversity in C. perspicillata . This disagrees with findings from neotropical bats captured in Mexico [ 108 ] and Belize [ 109 ], and a temperate insectivorous species [ 92 ]. Yet, our conclusions are drawn from a much larger sample size and greater within landscape replication. Still, how might we explain these contrasting observations? Bats are much more mobile and airborne compared to other mammals with detectable land-use effects on the microbiome [ 110 – 112 ]. In other words, bats may roost elsewhere but coalesce were food resources are plenty though with brief time spend in human-modified habitats [ 62 , 113 , 114 ]. Additionally, for diet generalists finding distinct microbiomes along a disturbance gradient may actually reflect shifts diet composition [ 18 , 115 – 117 ]. This would explain why the gut microbiome of the diet generalist frugivore A. jamaicensis was different in pristine forest sites but similar in urban and agricultural sites in Mexico [ 108 ]. In diet specialists, such as C. perspicillata , one of two scenarios are likely to follow land-use changes: either the species cannot find their preferred diet, declines in abundance and does simply not persist in fragmented and urbanized landscapes (as seen in many insectivorous bats [ 20 , 21 , 57 ]) or the species can find their preferred diet, thrives but their microbial composition remains unchanged. Our results fit squarely within the One Health framework by demonstrating how environmental change, disease ecology, and wildlife health intersect. Landscape differences affected the species in so far as to increase the likelihood of being infected in forest fragments surrounded by agricultural matrix. The change in gut microbial composition from AstV infection is therefore more likely in forest fragments. The loss of beneficial and enrichment of pathogenic members may shift the gut microbial community in ways relevant to host health and transmission. This highlights the importance of recognizing species- and context-specific dynamics when assessing disease risks within a One Health framework. Declarations Acknowledgments We thank the Smithsonian Tropical Research Institute in Panamá for providing excellent infrastructure and Oris Acevedo and Belkys Jimenéz for their help during fieldwork. We are grateful to all field assistants helping during mist-netting. We thank Ulrike Stehle for their technical support and Randall Jiménez, Luis Víquez, Ana Sofia Carranco and Marianella Mata Retana for laboratory assistance. Funding declaration This research was funded by the German Science Foundation (DFG) and is part of the DFG Priority Program SPP 1596: Ecology and species barriers in emerging infectious diseases (TS 81/7-1, SO 428/ 9-1, 9-2). Clinical trial number: not applicable Ethics, Consent to Participate, and Consent to Publish declarations: Capture and sampling techniques followed Panamanian protocols (MiAmbiente, Republica de Panama: SE/A-75-13 to SE/A-28-17) and were ethically certified by the Smithsonian Tropical Research Institute (IACUC protocols: 2014-0101-2016 to 2016-0627-2019). Data availability statement Sequencing data of 169 C. perspicillata can be accessed via the NCBI BioProject PNJNA715730 and the additional samples will be uploaded upon acceptance. Helicobacter sequences will be uploaded to NCBI upon acceptance. Statistical analysis and phyloseq object including metadata can be found under https://github.com/DominikWSchmid/Cperspicillata_AstV_16S. References Bosch A, Pintó RM, Guix S. Human astroviruses. Clin Microbiol Rev. 2014;27:1048–74. https://doi.org/10.1128/CMR.00013-14 . Wohlgemuth N, Honce R, Schultz-Cherry S. Astrovirus evolution and emergence. 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Supplementary Files SuppTable1.csv SuppTable2181225.csv SuppTable3.csv SuppTable4.csv SupplementaryFigure.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 13 May, 2026 Reviews received at journal 17 Apr, 2026 Reviews received at journal 14 Apr, 2026 Reviewers agreed at journal 06 Apr, 2026 Reviewers agreed at journal 24 Feb, 2026 Reviews received at journal 21 Feb, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviewers agreed at journal 07 Feb, 2026 Reviewers invited by journal 21 Jan, 2026 Editor invited by journal 20 Jan, 2026 Editor assigned by journal 26 Dec, 2025 Submission checks completed at journal 26 Dec, 2025 First submitted to journal 21 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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09:40:39","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":229177,"visible":true,"origin":"","legend":"","description":"","filename":"23068a0a711c43fdb448e6244e8414ce1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8419449/v1/2a5b7608f3e34d5dbb72089a.xml"},{"id":100984975,"identity":"1a89fad3-60e1-434b-8746-5f74429f2580","added_by":"auto","created_at":"2026-01-23 12:59:26","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":251602,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8419449/v1/8f961b2f5549f1b600598ae3.html"},{"id":100984961,"identity":"62881aa5-476e-4e1c-93b8-ba7864513252","added_by":"auto","created_at":"2026-01-23 12:59:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":364288,"visible":true,"origin":"","legend":"\u003cp\u003eA)\u003cstrong\u003e \u003c/strong\u003eImage of the Neotropical frugivore \u003cem\u003eCarollia perspicillata\u003c/em\u003e (credit: Dr. Thomas Hiller); B) Mean AstV prevalence (± standard error) captured either in continuous forest sites or forest fragments surrounded by agricultural matrix, and C) sample sizes for the subset for which the gut microbiome was sequenced after rarefying to 5.000 reads (AstV-negative = blue; AstV-positive = red).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8419449/v1/48a5025734609b5fddcccda3.png"},{"id":100984962,"identity":"9481268d-ee2b-445d-aeb0-c986ed7b2a53","added_by":"auto","created_at":"2026-01-23 12:59:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":154517,"visible":true,"origin":"","legend":"\u003cp\u003eA) Gut microbial composition and B) alpha-diversity (Shannon Diversity Index) by Astrovirus infection status (AstV-negative = blue; AstV-positive = red) and landscape. Bacterial genera contributing less than 1% of reads are grouped as ‘Other’. In the bean plot, the dashed line denotes the overall median, the longer solid line indicates group median, and shorter solid lines depict individual samples.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8419449/v1/ef7f7ff1ad2b490df27ef481.png"},{"id":100984967,"identity":"d68573a8-e6f5-4201-b492-45c08f832289","added_by":"auto","created_at":"2026-01-23 12:59:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":184082,"visible":true,"origin":"","legend":"\u003cp\u003eA) Principal coordinate analysis of the gut microbial beta-diversity (calculated as Aitchson distance from microbial abundances agglomerated to genus) and B-C) distances to centroid in relation to infection status or landscape. Dots are colored by infection status (AstV-negative = blue; AstV-positive = red) and statistically significant centroid differences are depicted as diamonds.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8419449/v1/18bf44b29545655296ee3fd4.png"},{"id":101203263,"identity":"5923b80c-9831-454d-84e1-92a412dc1964","added_by":"auto","created_at":"2026-01-27 09:39:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":308912,"visible":true,"origin":"","legend":"\u003cp\u003eA) Differential abundance plot depicting mean relative abundances and log fold changes in bacterial genera (prevalence \u0026gt; 0.1) in uninfected versus AstV-positive \u003cem\u003eC. perspicillata. \u003c/em\u003eB) phylogenetic relationship of the two \u003cem\u003eHelicobacter\u003c/em\u003e haplotypes (blue) identified in \u003cem\u003eC.\u003c/em\u003e \u003cem\u003eperspicillata\u003c/em\u003e relation to other known \u003cem\u003eHelicobacter\u003c/em\u003e species (GenBank accession numbers upon publication).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8419449/v1/0c992c3a98ec2a409ead7d63.png"},{"id":101208944,"identity":"c6c28d87-8e03-4904-a497-bd6b05263474","added_by":"auto","created_at":"2026-01-27 10:12:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1821636,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8419449/v1/bd3ffa44-fe66-486c-9532-33f48e9b0803.pdf"},{"id":101203304,"identity":"e31f69a8-0fb3-4688-8de6-96efc1a77c49","added_by":"auto","created_at":"2026-01-27 09:39:21","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1668,"visible":true,"origin":"","legend":"","description":"","filename":"SuppTable1.csv","url":"https://assets-eu.researchsquare.com/files/rs-8419449/v1/26a827a48257e1ceef340aee.csv"},{"id":100984966,"identity":"37c01252-8117-45ec-8e3c-a7e5b527fa8d","added_by":"auto","created_at":"2026-01-23 12:59:25","extension":"csv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9763,"visible":true,"origin":"","legend":"","description":"","filename":"SuppTable2181225.csv","url":"https://assets-eu.researchsquare.com/files/rs-8419449/v1/613aa81ee5d469413b9b34c5.csv"},{"id":100984963,"identity":"cc10b718-d7fa-4c8a-9c65-12a5c8fb0d0f","added_by":"auto","created_at":"2026-01-23 12:59:25","extension":"csv","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9557,"visible":true,"origin":"","legend":"","description":"","filename":"SuppTable3.csv","url":"https://assets-eu.researchsquare.com/files/rs-8419449/v1/41171044c58b0a7d4af24edd.csv"},{"id":101203632,"identity":"8e2a7c3e-5ad6-45c7-a259-c15af4735830","added_by":"auto","created_at":"2026-01-27 09:40:16","extension":"csv","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11671,"visible":true,"origin":"","legend":"","description":"","filename":"SuppTable4.csv","url":"https://assets-eu.researchsquare.com/files/rs-8419449/v1/fde05b7e06d8d731d72737e0.csv"},{"id":101203635,"identity":"643366bd-01ce-4d76-ad34-9161a181a30b","added_by":"auto","created_at":"2026-01-27 09:40:16","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":350129,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-8419449/v1/d2e7673a2a20721c090f8a4d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gut microbial composition changes in an Astrovirus-infected Neotropical frugivorous bat – a one health perspective","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAstroviruses (AstVs) are positive-sense, single-stranded enteric RNA viruses with an incredibly broad host range across diverse avian and mammalian hosts, including humans, and are directly and easily transmitted via the fecal-oral route [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The recent discovery of numerous novel astrovirus strains, their large genetic diversity and ability for cross-species recombination, highlight the risk of interspecies or even zoonotic transmission of AstVs [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In wildlife, AstV infections often persist asymptomatically, whereas in humans, infections can cause severe diarrhea and may lead to neuropathological encephalitis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Symptoms of AstV infection vary depending on age and immunological status of infected hosts with children and immunosuppressed being most affected [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLand-use changes and, as a consequence, increased likelihood of human-wildlife contacts amplify the chances of disease spread beyond species boundaries [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Especially, food and nutrient scarcity in man-made landscapes increases overlap between wildlife, domesticated animals and humans [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Prominent examples include the regular spillover events of the zoonotic Nipah and Hendra viruses from fruit bats to pigs and horses [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and the repeated transmission of morbilli or lyssaviruses from vampire bats to pigs and cattle [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The high degree of population fragmentation in mosaic landscapes and the frequent crossover with novel hosts may even accelerate virus evolution, exacerbating their zoonotic risk [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Besides, the health consequences for wildlife inhabiting fragmented forests, agricultural landscapes or urban environments are often poorly understood or entirely unknown [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe negative health effects from enteropathogenic viral infections, such as those caused by AstVs, may arise from cascading effects on the host\u0026rsquo;s gastrointestinal microbiome, enhancing viral activity or facilitating co-infections (e.g., norovirus [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], rotavirus [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], coronavirus [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], adenovirus [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and astrovirus [\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]). The gastrointestinal microbiome is an incredibly complex and highly diverse ecosystem, with important roles in nutrient acquisition (e.g., vitamins; [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]), toxin degradation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and metabolism [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] as well as in the defense against enteropathogens [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and the development and maintenance of immune responses [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Abnormal shifts in its diversity and composition, beyond the normal range of intraspecific variation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], could be either cause or consequence of the susceptibility to pathogens or chronic inflammatory diseases [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. For instance, increased viral load and co-infections amplify gut microbial dysbiosis in bats and voles [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, the interaction between viruses and the hosts\u0026rsquo; microbiome is an essential, yet often overlooked, component in the holobiont machinery [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAstroviruses are the fourth most common virus family recovered from bats, and a high diversity of AstVs have been reported in bat species worldwide [\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Due to their high prevalence and presumably mild pathogenicity AstVs represent a perfect study system to understand potential health effects in visibly symptomless bats. At the same time, bats are seen as a model system to investigate the role of microbes in host evolution, physiology and fitness as they harbor a diverse set of microbiomes likely reflecting their ecological and phylogenetic diversity ([\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] but see [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and discussion in [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]). Unsurprising therefore, that a previous study identified AstV-mediated disruption of the gut microbial community in the Jamaican fruit bat (\u003cem\u003eArtibeus jamaicensis\u003c/em\u003e) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Little is, however, known about the health consequences of land-use changes for bats [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], and the role of disease ecology in that context. From a OneHealth perspective, a better understanding of how land-use change, host microbial health and infection ecology interact is pivotal to predict and prevent spillover events, and safeguard human and animal wellbeing [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we investigated the effect and interplay of AstV infection and land-use change on the gut microbiome diversity and composition in a neotropical frugivorous habitat generalist, the Seba\u0026acute;s short-tailed bat (\u003cem\u003eCarollia perspicillata\u003c/em\u003e). The species is abundant throughout Central and South America, often profiting from disturbed areas and the abundance of fruits of early succession, hence found in human-modified habitats, even often roosting close to rural settlements [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. As the microbiome is the first line of defense in the gut and linked to physiological stress responses, we hypothesized that AstV-infections and human disturbance reduce microbial alpha diversity, shift microbial beta diversity, and favor the emergence of (opportunistically) pathogenic bacteria. Age may also play a role, as shown in \u003cem\u003eA. jamaicensis\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], immunologically na\u0026iuml;ve subadults exhibited altered gut microbiota when infected with AstVs. Such age-dependent responses may therefore be common in bats. Moreover, we predict that the effect on the microbiota is amplified in AstV-positive bats inhabiting forest fragments embedded in an agricultural matrix.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFocus species and fecal sample collection\u003c/h2\u003e \u003cp\u003eThe phyllostomid bat \u003cem\u003eC. perspicillata\u003c/em\u003e is a widespread frugivore (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), found in high abundance across large parts of Central and South America [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. \u003cem\u003eC. perspicillata\u003c/em\u003e consumes fruits of early successional plants, e.g., pepper plants (genus: \u003cem\u003ePiper\u003c/em\u003e) [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The species shows flexible roosting behavior in crevices and hollows, while flight capabilities limit the species to searching for food in relative proximity to its roost (mean: 1.6 km) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Its diet preference for fruits from successional plants found frequently in agricultural landscapes cleared for livestock does not only allows the species to persist but even to thrive in human-disturbed habitats [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBats were caught in lowland tropical rainforest ecosystems of Central Panama using mist-nets (ECOTONE, Gdynia, Poland) in a standardized sampling design at five independent sampling sites in three landscapes varying in degree of fragmentation and human disturbance: natural lowland forest, forest fragments embedded in an agricultural matrix, and forested islands surrounded by water [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. However, \u003cem\u003eC. perspicillata\u003c/em\u003e is rarely found on islands [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Thus, the work here only concerns sites in old-growth forest and forest fragments surrounded by agricultural landscapes. After capture, individuals were sexed, and age was determined based on the ossification of the epiphyses of the digits [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Individual fecal samples were collected directly from bats upon defecation during handling or collected out of the fabric bags, in which bats were kept temporarily before processing, and immediately preserved in RNAlater (Life Technologies). All bats were released on site right after handling. Capture and sampling techniques followed Panamanian protocols (MiAmbiente, Republica de Panama: SE/A-75-13 to SE/A-28-17) and were ethically certified by the Smithsonian Tropical Research Institute (IACUC protocols: 2014-0101-2016 to 2016-0627-2019).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAstrovirus testing\u003c/h3\u003e\n\u003cp\u003eViral RNA was extracted from 434 fecal samples using the MagNA Pure 96 DNA and the Viral NA Small Volume Kit (Roche) corresponding to the manufacturer\u0026rsquo;s guidelines. AstV prevalence was determined by the broadly reactive nested reverse transcription-PCR (RT-PCR) assay, as described previously [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eBacterial DNA extraction and 16S rRNA gene bacterial amplicon sequencing\u003c/h3\u003e\n\u003cp\u003eMicrobiome analyses were carried out in a total of 234 individuals. After homogenization of fecal samples (SpeedMill PLUS Homogenizer, Analytik Jena, Germany), bacterial genomic DNA was extracted applying the NucleoSpin 96 Soil kit (Macherey-Nagel, Germany) following the manufacturer's instructions. The universal 16S primers 515F/806R were used for PCR amplification of the hypervariable V4 region (291 bp) of 16S rRNA \u0026ndash; a standard primer pair, which leads to very high bacterial diversity and performed best in a recent primer comparison experiment [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. We used the Fluidigm System (Access Array\u0026trade; System for Illumina Sequencing Systems, \u0026copy;Fluidigm Corporation) for primer tagging. PCRs (15 \u0026micro;l reaction volume) were executed as described previously [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. After purification (NucleoMag bead-based size selection, Macherey-Nagel, Germany) and quantification of barcoded samples (DropSense, Trinean, US), we applied paired-end sequencing on the Illumina\u0026reg; MiSeq platform.\u003c/p\u003e\n\u003ch3\u003eBioinformatics\u003c/h3\u003e\n\u003cp\u003eAll subsequent analyses were performed within R (v4.4.1, [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]). We applied the \u003cem\u003eDADA2\u003c/em\u003e pipeline [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Reads were trimmed from both ends to remove low-quality regions, and after denoising and merging, chimeric sequences were removed, following the \u0026lsquo;consensus\u0026rsquo; method as implemented in \u003cem\u003eDADA2\u003c/em\u003e. A na\u0026iuml;ve Bayesian classifier within SILVA v138 database was applied for taxonomic assignments of obtained nonchimeric amplicon sequence variants (ASVs). The \u003cem\u003ephyloseq\u003c/em\u003e package (v1.48, [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]) was further used for data processing. ASVs assigned to chloroplasts, mitochondria, and unassigned ASVs at the phylum level were removed from the dataset, and cleaned using the \u003cem\u003eMicroViz\u003c/em\u003e package (v0.12.5; [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]).\u003c/p\u003e\n\u003ch3\u003eAstrovirus prevalence\u003c/h3\u003e\n\u003cp\u003eFirst, we calculated the average prevalence of AstV among 434 virus-screened \u003cem\u003eC. perspicillata\u003c/em\u003e for each sampling site. We compared mean prevalence (square-root transformed to meet normality assumption) using one-sided t-test with landscape (continuous forests vs. forested fragments in an agricultural matrix) as explanatory variable.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGut microbial composition and alpha-diversity\u003c/h2\u003e \u003cp\u003eWe calculated the gut microbial alpha diversity indices, the number of observed ASVs, Shannon index and Faith\u0026rsquo;s phylogenetic diversity, after rarefying the data to 5000 sequences per sample. Rarefying removed 35 samples with fewer than 5000 reads (final sample size: n\u0026thinsp;=\u0026thinsp;199; [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]), although unrarefied data yielded comparable results (Supplementary Fig.\u0026nbsp;1). To analyze the effects on alpha diversity metrics (Faith\u0026rsquo;s PD and Observed ASVs were log-transformed to meet normality assumptions), we used linear mixed-effects models (LMERs) using the \u003cem\u003elme4\u003c/em\u003e package (v1.1-37; [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]) including AstV-infection (positive vs. negative), age (subadults vs. adults), sex (male vs. female), season (wet vs. dry), and landscape (continuous forests vs. forested fragments in an agricultural matrix) as explanatory variables, while still accounting for sequencing depth and setting sampling site as random effect. Since a previous study revealed that AstV infections influenced the gut microbiome dependent on host age [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and we aimed to unveil potential interactions between AstV-infection and landscape differences, we included both two-way interactions in each model, and back-selected.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGut microbial beta-diversity\u003c/h3\u003e\n\u003cp\u003eMicrobial beta diversity distances (i.e., Bray-Curtis, Jaccard, Aitchison, weighted and unweighted Unifrac) were calculated for ASVs and ASVs agglomerated at genus (since higher level taxonomic assignments are often more reliable for wildlife 16S data). The gut microbial dissimilarity was compared using permutational analyses of variance (999 permutations) encoded in the \u003cem\u003evegan\u003c/em\u003e package (v2.7-2; [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]). We used the same explanatory variables as for the LMERs and stratified by sampling site. Principal coordinate analyses (PCoA) were performed to visualize the pattern of separation between the various categories of samples.\u003c/p\u003e\n\u003ch3\u003eDifferential abundance analysis\u003c/h3\u003e\n\u003cp\u003eFinally, we performed an analysis of compositions of microbiomes with bias correction at ASV and genus levels using both AstV infection status and landscape as explanatory factors [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. To avoid spurious results, we removed ASVs or genera prevalent in fewer than 10% of samples, as recommended [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e], and applied false-discovery rate corrections [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAstrovirus prevalence and microbiome composition\u003c/h2\u003e \u003cp\u003eThe prevalence of AstV infections was higher in the forest fragments surrounded by agricultural matrix (mean 22.8% \u0026plusmn; 1.6 SE; t-value\u0026thinsp;=\u0026thinsp;4.3, p\u0026thinsp;=\u0026thinsp;0.002; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) than in the continuous forest (mean 10.1% \u0026plusmn; 2.5 standard error). After initial filtering, 7.188.339 sequence reads of the V4 region the bacterial 16S ribosomal RNA gene remained from 234 fecal samples of \u003cem\u003eC. perspicillata\u003c/em\u003e. On average 17.575 (range: 1.945\u0026ndash;110.054) high-quality reads per sample were obtained after taxonomic assignments. Rarefying to a representative number of 5.000 reads resulted in the removal of 35 samples, leaving 137 AstV-negative and 62 AstV-positive \u003cem\u003eC. perspicillata\u003c/em\u003e for further analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe dominant phylum was Pseudomonadota (mean 49.9% \u0026plusmn; 28.9 standard deviation). At family resolution Enterobacteriaceae (mean 21.5% \u0026plusmn; 29.4 SD) made up the highest proportion, followed by Mycoplasmataceae (mean 10.7% \u0026plusmn; 22.1 SD) and Streptococcaceae (mean 10.1% \u0026plusmn; 15.4 SD) of the microbial community (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffects of AstV infection and landscape on alpha and beta diversity\u003c/h2\u003e \u003cp\u003eNeither landscape differences nor infection status significantly affected any of the alpha diversity in \u003cem\u003eC. perspicillata\u003c/em\u003e (Supp. Table\u0026nbsp;1). There was a very weak tendency for a higher Shannon value in \u003cem\u003eC. perispicillata\u003c/em\u003e positive for AstV in continuous forest, while lower values for AstV-positive individuals were recovered among the forest fragments (infection-landscape interaction: F\u003csub\u003e1,191\u003c/sub\u003e=2.83, p\u0026thinsp;=\u0026thinsp;0.094; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Neither the interaction nor single effect of infection or landscape was found in any other alpha diversity metric (Supp. Table\u0026nbsp;1). Subadult \u003cem\u003eC. perispicillata\u003c/em\u003e had a lower Shannon diversity (F\u003csub\u003e1,191\u003c/sub\u003e=4.52, p\u0026thinsp;=\u0026thinsp;0.034). Observed ASV richness increased with sequencing depth in spite of rarefaction (Supp. Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eAstV infection shifted the beta diversity centroid in all distances except for weighted Unifrac (Supp. Table\u0026nbsp;2). This implies that the infection substantially affecting microbial community composition and, to a lesser extent, structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A weak landscape effect was only found in Aitchison distance calculated from ASV matrix (R2\u0026thinsp;=\u0026thinsp;0.010; p\u0026thinsp;=\u0026thinsp;0.023), but the interaction between AstV infection and landscape was never significant. Sequencing depth affected particularly distances that weighted microbial abundances based on reads (Supp. Table\u0026nbsp;2). Beta dispersion did not differ between infection status (F\u003csub\u003e1,197\u003c/sub\u003e=0.14, p\u0026thinsp;=\u0026thinsp;0.246; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) or landscape (F\u003csub\u003e1,197\u003c/sub\u003e=0.07, p\u0026thinsp;=\u0026thinsp;0.780; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAstV infection is associated with shifts in the relative abundance of genera and ASVs\u003c/h2\u003e \u003cp\u003eIn total, 29 ASVs and 23 genera were found to be differentially abundant between AstV-positive and AstV-negative C. \u003cem\u003eperspicillata\u003c/em\u003e (Supp. Table\u0026nbsp;3, 4): The genera \u003cem\u003eHelicobacter\u003c/em\u003e, \u003cem\u003eLeptotrichia\u003c/em\u003e, \u003cem\u003eNeisseria\u003c/em\u003e and \u003cem\u003eMoraxella\u003c/em\u003e, genera containing species pathogenic to humans or animals, were enriched in AstV-positive \u003cem\u003eC. perspicillata\u003c/em\u003e, for instance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Based on these results, we amplified an approximately 1200 bp long sequence in 40 randomly selected samples per landscape using a \u003cem\u003eHelicobacter\u003c/em\u003e-specific primer pair [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e], and aligned the resulting sequences to sequences from 27 \u003cem\u003eHelicobacter\u003c/em\u003e species and \u003cem\u003eWolinella succinogenes\u003c/em\u003e as outgroup (see Supplementary Material for more details). Two \u003cem\u003eHelicobacter\u003c/em\u003e haplotypes (\u003cem\u003eC.per\u003c/em\u003e_haplotype 1 and \u003cem\u003eC.per\u003c/em\u003e_haplotype 2) were identified and aligned most closely to \u003cem\u003eHelicobacter anseris\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eBy contrast, bacterial genera often beneficial to hosts, such the lactic-acid producing \u003cem\u003eLactococcus\u003c/em\u003e, the sugar-fermenting order of Saccharimonadales, aromatic compound degrading genus of \u003cem\u003eNovosphingobium\u003c/em\u003e, decreased in abundance in AstV-positive bats. Although landscape effects were minimal on the whole community, certain bacteria were still recovered more abundant in continuous forests than forest fragments independent of the infection status (e.g., \u003cem\u003ePaenibacillus\u003c/em\u003e and \u003cem\u003eActinomyces\u003c/em\u003e; Supp. Table\u0026nbsp;3, 4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBats are known to host numerous virus families with zoonotic potential [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and harbor a particularly high diversity of astroviruses, playing a key role in AstV ecology and evolution [\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Seba\u0026rsquo;s short-tailed bats (\u003cem\u003eC. perspicillata\u003c/em\u003e) appear healthy and show no overt clinical symptoms (e.g., weakness), yet they were found to be infected with astroviruses. Astrovirus prevalence was higher in bats captured in forest fragments than in old-growth lowland rainforest sites in Panama. Furthermore, gut microbial community composition\u0026mdash;a proxy for host health [\u003cspan additionalcitationids=\"CR81\" citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]\u0026mdash;differed between AstV-negative and AstV-positive bats, suggesting that enteropathogenic viral infection influences both the taxonomic and, in all likelihood, functional diversity of the microbiome. A decline in gut microbial homeostasis during infection may increase the risk of co-infection [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and enhance viral (and bacterial) shedding [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e], posing a potential One Health concern. Consistent with this, two novel \u003cem\u003eHelicobacter\u003c/em\u003e haplotypes closely related to \u003cem\u003eH. anseris\u003c/em\u003e were detected in AstV-positive \u003cem\u003eC. perspicillata\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eHuman encroachment into nature is likely the main driver behind the current extinction crisis [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Additionally, research over the last decades has shown that land-use changes further increase the risk of disease spillover from wild animals into livestock and humans [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. We document that AstVs were twice as likely to be detected in \u003cem\u003eC. perspicillata\u003c/em\u003e captured in forest fragments embedded in agricultural landscapes than in individuals captured in continuous forests. This is unlike findings from the Bornean rainforest where AstV infection likelihood in insectivorous bats captured in actively logged, fragmented and recovering forest sites did not differ [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, landscape differences affected neither AstV prevalence [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] nor host abundance of the canopy frugivore \u003cem\u003eA. jamaicensis\u003c/em\u003e [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. By contrast, the understory frugivore \u003cem\u003eC. perspicillata\u003c/em\u003e, which consumes fruits of early successional plants [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], such as the pepper plants (genus \u003cem\u003ePiper\u003c/em\u003e) \u0026ndash; common in edge habitat surrounding forest fragments \u0026ndash;, is four times more likely to be captured in forest fragments than continuous forest sites [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Our finding lends weight to the idea that resource availability drives spatial host densities, and that viral transmission is thus largely density mediated [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. However, land-use change is still at the root of the rise in disease prevalence, because regionally patchy resources become more localized, amassing a higher number of hosts and, thus, increasing transmission risk.\u003c/p\u003e \u003cp\u003eThe gut microbial community of AstV-positive \u003cem\u003eC. perspicillata\u003c/em\u003e was shifted independently of whether the bat was captured in forest fragments or old growth forest sites. This implies land-use differences do not amplify disease-mediated changes to the gut microbial community. The gut microbial community was also different in \u003cem\u003eA. jamaicensis\u003c/em\u003e infected with AstVs although in this case the effect was dependent on host age [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. While cause and effect are difficult to disentangle in naturally infected populations [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e], AstV infections are known to alter the gut mucus barrier [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], offering a mechanism by which an enteropathogenic virus could tamper with the balance of beneficial and pathogenic gut bacteria [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. Indeed, some beneficial bacteria, i.e., the lactic acid-producing genus \u003cem\u003eLactococcus\u003c/em\u003e, adhere to the outer mucus layer surrounding host epithelial cells using pili, anchoring and mucin-binding proteins [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. A disruption of the mucus barrier may therefore have consequences for resident symbionts. While we cannot elucidate the precise mechanism, \u003cem\u003eLactococcus\u003c/em\u003e and other potentially beneficial gut bacteria, such as the aromatic compound degrading genus of \u003cem\u003eNovosphingobium\u003c/em\u003e \u0026ndash; identified to support the breakdown of defensive plant flavonoids [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e] \u0026ndash; declined in AstV-positive bats.\u003c/p\u003e \u003cp\u003eBacterial ASVs or genera that increased in AstV-positive bats range from commensals to pathogens, including the genera \u003cem\u003eLeptotrichia\u003c/em\u003e and \u003cem\u003eHelicobacter\u003c/em\u003e. Many \u003cem\u003eLeptotrichia\u003c/em\u003e are able to ferment mono- and disaccharides to lactic acid and are found as commensal part in healthy hosts\u0026rsquo; oral and intestinal microbiome [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. Yet, \u003cem\u003eLeptotrichia\u003c/em\u003e can be opportunistically pathogenic and was implicated in dental decay [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e], or gut dysbiosis in hosts with bacterial or viral co-infections [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e, \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e]. The bacterial genus most strongly enriched in AstV-positive bats was \u003cem\u003eHelicobacter\u003c/em\u003e, however. \u003cem\u003eHelicobacter\u003c/em\u003e are gram-negative bacteria able to penetrate the mucous lining of the gut due to their helical and flagellated morphology [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e]. In humans, infections with \u003cem\u003eHelicobacter pylori\u003c/em\u003e cause gastric disorders even inducing cancer [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. Other \u003cem\u003eHelicobacter\u003c/em\u003e species circulate in livestock and wildlife, and are often associated with gastric histopathological alterations [\u003cspan additionalcitationids=\"CR100\" citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e]. We identified \u003cem\u003eHelicobacter\u003c/em\u003e haplotypes clustering most closely to \u003cem\u003eHelicobacter anseris\u003c/em\u003e, which was described in Canada geese for the first time in 2006 [\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e], but has since been isolated in a number of birds from South America [\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e, \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e]. \u003cem\u003eH. anseris\u003c/em\u003e grows optimally in temperatures ranging between 37 and 42\u0026deg;C [\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e], which makes it feasible that \u003cem\u003eH. anseris\u003c/em\u003e or a closely related strain could thrive in bats, which similar to birds maintain elevated body temperatures during flight [\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e, \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e]. In addition, bats and birds share similar gut microbial communities, which are unlike those of other mammals [\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e]. One could theorise that Helicobacter strains evolved in birds can infect host with a similar gut microbial community more easily. Yet, we lack histopathological information to determine if the \u003cem\u003eHelicobacter\u003c/em\u003e haplotypes were harmful to \u003cem\u003eC. perspicillata\u003c/em\u003e. Taken together, AstV infection in bats seem to replace beneficial bacteria with potentially pathogenic ones, undermining the services the host may derive from its microbiota.\u003c/p\u003e \u003cp\u003eIn comparison with the AstV infection, land-use differences seem to matter little to the gut microbial alpha and beta diversity in \u003cem\u003eC. perspicillata\u003c/em\u003e. This disagrees with findings from neotropical bats captured in Mexico [\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e] and Belize [\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e], and a temperate insectivorous species [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. Yet, our conclusions are drawn from a much larger sample size and greater within landscape replication. Still, how might we explain these contrasting observations? Bats are much more mobile and airborne compared to other mammals with detectable land-use effects on the microbiome [\u003cspan additionalcitationids=\"CR111\" citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e]. In other words, bats may roost elsewhere but coalesce were food resources are plenty though with brief time spend in human-modified habitats [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e, \u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e]. Additionally, for diet generalists finding distinct microbiomes along a disturbance gradient may actually reflect shifts diet composition [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR116\" citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e]. This would explain why the gut microbiome of the diet generalist frugivore \u003cem\u003eA. jamaicensis\u003c/em\u003e was different in pristine forest sites but similar in urban and agricultural sites in Mexico [\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e]. In diet specialists, such as \u003cem\u003eC. perspicillata\u003c/em\u003e, one of two scenarios are likely to follow land-use changes: either the species cannot find their preferred diet, declines in abundance and does simply not persist in fragmented and urbanized landscapes (as seen in many insectivorous bats [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]) or the species can find their preferred diet, thrives but their microbial composition remains unchanged.\u003c/p\u003e \u003cp\u003eOur results fit squarely within the One Health framework by demonstrating how environmental change, disease ecology, and wildlife health intersect. Landscape differences affected the species in so far as to increase the likelihood of being infected in forest fragments surrounded by agricultural matrix. The change in gut microbial composition from AstV infection is therefore more likely in forest fragments. The loss of beneficial and enrichment of pathogenic members may shift the gut microbial community in ways relevant to host health and transmission. This highlights the importance of recognizing species- and context-specific dynamics when assessing disease risks within a One Health framework.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Smithsonian Tropical Research Institute in Panam\u0026aacute; for providing excellent infrastructure and Oris Acevedo and Belkys Jimen\u0026eacute;z for their help during fieldwork. We are grateful to all field assistants helping during mist-netting. We thank Ulrike Stehle for their technical support and Randall Jim\u0026eacute;nez, Luis V\u0026iacute;quez, Ana Sofia Carranco and Marianella Mata Retana for laboratory assistance. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the German Science Foundation (DFG) and is part of the DFG Priority Program SPP 1596: Ecology and species barriers in emerging infectious diseases (TS 81/7-1, SO 428/ 9-1, 9-2).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e not applicable\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics, Consent to Participate, and Consent to Publish declarations:\u003c/strong\u003e Capture and sampling techniques followed Panamanian protocols (MiAmbiente, Republica de Panama: SE/A-75-13 to SE/A-28-17) and were ethically certified by the Smithsonian Tropical Research Institute (IACUC protocols: 2014-0101-2016 to 2016-0627-2019).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability statement \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequencing data of 169 \u003cem\u003eC. perspicillata \u003c/em\u003ecan be accessed via the NCBI BioProject PNJNA715730 and the additional samples will be uploaded upon acceptance. Helicobacter sequences will be uploaded to NCBI upon acceptance. Statistical analysis and phyloseq object including metadata can be found under https://github.com/DominikWSchmid/Cperspicillata_AstV_16S. \u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBosch A, Pint\u0026oacute; RM, Guix S. Human astroviruses. Clin Microbiol Rev. 2014;27:1048\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/CMR.00013-14\u003c/span\u003e\u003cspan address=\"10.1128/CMR.00013-14\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWohlgemuth N, Honce R, Schultz-Cherry S. Astrovirus evolution and emergence. 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Mol Ecol. 2025;34:e17782. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/mec.17782\u003c/span\u003e\u003cspan address=\"10.1111/mec.17782\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8419449/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8419449/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAstroviruses are becoming a growing concern in public and veterinary health. In humans, astrovirus infections can cause severe diarrhea and may lead to neuropathological encephalitis, whereas in wildlife, these enteropathogenic viral infections often lack overt symptoms and, thus, remain unnoticed. Yet, their close interaction with the host\u0026rsquo;s gastrointestinal microbiome might drive cascading effects with disadvantages for host health. Bats harbor many zoonotic viruses without showing signs of disease, and many species move freely along the gradient from pristine to agricultural landscapes. To better understand the impact of Astrovirus (AstV) infection under a One Health framework, we investigated the gut microbiome of naturally AstV-infected Seba\u0026rsquo;s short-tailed bats (\u003cem\u003eCarollia perspicillata\u003c/em\u003e; n\u0026thinsp;=\u0026thinsp;234) inhabiting old-growth lowland forests or forest fragments embedded in an agricultural matrix in Panama. AstV prevalence was higher in forest fragments. We observed that AstV infection is associated with a shift in microbial beta but not alpha diversity, which points towards the replacement of common gut microbial taxa when infected. Indeed, potential beneficial bacteria, such as \u003cem\u003eLactococcus\u003c/em\u003e, decreased in abundance, whereas potentially pathogenic bacteria from the \u003cem\u003eHelicobacter\u003c/em\u003e genus increased in AstV-positive bats. Two \u003cem\u003eHelicobacter\u003c/em\u003e haplotypes closely related to avian \u003cem\u003eHelicobacter\u003c/em\u003e species were identified. We conclude that even though the impact of infection on the microbiome was not amplified in forest fragments, the higher infection likelihood in landscapes altered by humans implies more frequent or prolonged health repercussions for bats.\u003c/p\u003e","manuscriptTitle":"Gut microbial composition changes in an Astrovirus-infected Neotropical frugivorous bat – a one health perspective","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 12:59:20","doi":"10.21203/rs.3.rs-8419449/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-13T07:34:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-17T17:40:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-14T12:33:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79320917452502756676128104520548053273","date":"2026-04-06T16:29:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41439315313648419888174937815382438418","date":"2026-02-24T12:23:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-22T00:40:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"128881206752205049795913280763855378503","date":"2026-02-18T14:53:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"251838806048191107234942283039840929366","date":"2026-02-07T21:42:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-21T21:01:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-20T19:34:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-26T11:00:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-26T10:59:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-12-21T20:49:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6ab7cd30-6b8b-46ff-ab11-56a94e50fa84","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-13T07:34:55+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T07:44:25+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-23 12:59:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8419449","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8419449","identity":"rs-8419449","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}