A cryptic plasmid is among the most numerous genetic elements in the human gut.

Plasmids are extrachromosomal genetic elements that often encode fitness-enhancing features. However, many bacteria carry "cryptic" plasmids that do not confer clear beneficial functions. We identified one such cryptic plasmid, pBI143, which is ubiquitous across industrialized gut microbiomes and is 14 times as numerous as crAssphage, currently established as the most abundant extrachromosomal genetic element in the human gut. The majority of mutations in pBI143 accumulate in specific positions across thousands of metagenomes, indicating strong purifying selection. pBI143 is monoclonal in most individuals, likely due to the priority effect of the version first acquired, often from one's mother. pBI143 can transfer between Bacteroidales, and although it does not appear to impact bacterial host fitness in vivo, it can transiently acquire additional genetic content. We identified important practical applications of pBI143, including its use in identifying human fecal contamination and its potential as an alternative approach to track human colonic inflammatory states.

Beyond gut instincts: Microbe survival depends on sugars and butyrate.

The factors and mechanisms that shape the composition and function of closely related members in a complex microbial community are largely unknown. The study by Park and colleagues reveals that the fitness of various Bacteroidales species and strains in the gut microbiome is regulated by butyrate in a glycan-dependent manner.

Strain-level fitness in the gut microbiome is an emergent property of glycans and a single metabolite.

The human gut microbiota resides within a diverse chemical environment challenging our ability to understand the forces shaping this ecosystem. Here, we reveal that fitness of the Bacteroidales, the dominant order of bacteria in the human gut, is an emergent property of glycans and one specific metabolite, butyrate. Distinct sugars serve as strain-variable fitness switches activating context-dependent inhibitory functions of butyrate. Differential fitness effects of butyrate within the Bacteroides are mediated by species-level variation in Acyl-CoA thioesterase activity and nucleotide polymorphisms regulating an Acyl-CoA transferase. Using in vivo multi-omic profiles, we demonstrate Bacteroides fitness in the human gut is associated together, but not independently, with Acyl-CoA transferase expression and butyrate. Our data reveal that each strain of the Bacteroides exists within a unique fitness landscape based on the interaction of chemical components unpredictable by the effect of each part alone mediated by flexibility in the core genome.

Transient Osmotic Perturbation Causes Long-Term Alteration to the Gut Microbiota.

Osmotic diarrhea is a prevalent condition in humans caused by food intolerance, malabsorption, and widespread laxative use. Here, we assess the resilience of the gut ecosystem to osmotic perturbation at multiple length and timescales using mice as model hosts. Osmotic stress caused reproducible extinction of highly abundant taxa and expansion of less prevalent members in human and mouse microbiotas. Quantitative imaging revealed decimation of the mucus barrier during osmotic perturbation, followed by recovery. The immune system exhibited temporary changes in cytokine levels and a lasting IgG response against commensal bacteria. Increased osmolality prevented growth of commensal strains in vitro, revealing one mechanism contributing to extinction. Environmental availability of microbiota members mitigated extinction events, demonstrating how species reintroduction can affect community resilience. Our findings (1) demonstrate that even mild osmotic diarrhea can cause lasting changes to the microbiota and host and (2) lay the foundation for interventions that increase system-wide resilience.

From Hype to Hope: The Gut Microbiota in Enteric Infectious Disease.

One of the clearest functions of the gut microbiota in humans is resistance to colonization by enteric bacterial pathogens. Reconstitution of the microbiota offers an exciting therapeutic approach, but great challenges must be overcome.

Extensive strain-level copy-number variation across human gut microbiome species.

Within each bacterial species, different strains may vary in the set of genes they encode or in the copy number of these genes. Yet, taxonomic characterization of the human microbiota is often limited to the species level or to previously sequenced strains, and accordingly, the prevalence of intra-species variation, its functional role, and its relation to host health remain unclear. Here, we present a comprehensive large-scale analysis of intra-species copy-number variation in the gut microbiome, introducing a rigorous computational pipeline for detecting such variation directly from shotgun metagenomic data. We uncover a large set of variable genes in numerous species and demonstrate that this variation has significant functional and clinically relevant implications. We additionally infer intra-species compositional profiles, identifying population structure shifts and the presence of yet uncharacterized variants. Our results highlight the complex relationship between microbiome composition and functional capacity, linking metagenome-level compositional shifts to strain-level variation.

Periportal macrophages protect against commensal-driven liver inflammation.

The liver is the main gateway from the gut, and the unidirectional sinusoidal flow from portal to central veins constitutes heterogenous zones, including the periportal vein (PV) and the pericentral vein zones1-5. However, functional differences in the immune system in each zone remain poorly understood. Here intravital imaging revealed that inflammatory responses are suppressed in PV zones. Zone-specific single-cell transcriptomics detected a subset of immunosuppressive macrophages enriched in PV zones that express high levels of interleukin-10 and Marco, a scavenger receptor that sequesters pro-inflammatory pathogen-associated molecular patterns and damage-associated molecular patterns, and consequently suppress immune responses. Induction of Marco+ immunosuppressive macrophages depended on gut microbiota. In particular, a specific bacterial family, Odoribacteraceae, was identified to induce this macrophage subset through its postbiotic isoallolithocholic acid. Intestinal barrier leakage resulted in inflammation in PV zones, which was markedly augmented in Marco-deficient conditions. Chronic liver inflammatory diseases such as primary sclerosing cholangitis (PSC) and non-alcoholic steatohepatitis (NASH) showed decreased numbers of Marco+ macrophages. Functional ablation of Marco+ macrophages led to PSC-like inflammatory phenotypes related to colitis and exacerbated steatosis in NASH in animal experimental models. Collectively, commensal bacteria induce Marco+ immunosuppressive macrophages, which consequently limit excessive inflammation at the gateway of the liver. Failure of this self-limiting system promotes hepatic inflammatory disorders such as PSC and NASH.

Identification of trypsin-degrading commensals in the large intestine.

Increased levels of proteases, such as trypsin, in the distal intestine have been implicated in intestinal pathological conditions1-3. However, the players and mechanisms that underlie protease regulation in the intestinal lumen have remained unclear. Here we show that Paraprevotella strains isolated from the faecal microbiome of healthy human donors are potent trypsin-degrading commensals. Mechanistically, Paraprevotella recruit trypsin to the bacterial surface through type IX secretion system-dependent polysaccharide-anchoring proteins to promote trypsin autolysis. Paraprevotella colonization protects IgA from trypsin degradation and enhances the effectiveness of oral vaccines against Citrobacter rodentium. Moreover, Paraprevotella colonization inhibits lethal infection with murine hepatitis virus-2, a mouse coronavirus that is dependent on trypsin and trypsin-like proteases for entry into host cells4,5. Consistently, carriage of putative genes involved in trypsin degradation in the gut microbiome was associated with reduced severity of diarrhoea in patients with SARS-CoV-2 infection. Thus, trypsin-degrading commensal colonization may contribute to the maintenance of intestinal homeostasis and protection from pathogen infection.

NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis.

Inflammasomes are multiprotein complexes that function as sensors of endogenous or exogenous damage-associated molecular patterns. Here, we show that deficiency of NLRP6 in mouse colonic epithelial cells results in reduced IL-18 levels and altered fecal microbiota characterized by expanded representation of the bacterial phyla Bacteroidetes (Prevotellaceae) and TM7. NLRP6 inflammasome-deficient mice were characterized by spontaneous intestinal hyperplasia, inflammatory cell recruitment, and exacerbation of chemical colitis induced by exposure to dextran sodium sulfate (DSS). Cross-fostering and cohousing experiments revealed that the colitogenic activity of this microbiota is transferable to neonatal or adult wild-type mice, leading to exacerbation of DSS colitis via induction of the cytokine, CCL5. Antibiotic treatment and electron microscopy studies further supported the role of Prevotellaceae as a key representative of this microbiota-associated phenotype. Altogether, perturbations in this inflammasome pathway, including NLRP6, ASC, caspase-1, and IL-18, may constitute a predisposing or initiating event in some cases of human IBD.

Species invasions shift microbial phenology in a two-decade freshwater time series.

Invasive species impart abrupt changes on ecosystems, but their impacts on microbial communities are often overlooked. We paired a 20 y freshwater microbial community time series with zooplankton and phytoplankton counts, rich environmental data, and a 6 y cyanotoxin time series. We observed strong microbial phenological patterns that were disrupted by the invasions of spiny water flea (Bythotrephes cederströmii) and zebra mussels (Dreissena polymorpha). First, we detected shifts in Cyanobacteria phenology. After the spiny water flea invasion, Cyanobacteria dominance crept earlier into clearwater; and after the zebra mussel invasion, Cyanobacteria abundance crept even earlier into the diatom-dominated spring. During summer, the spiny water flea invasion sparked a cascade of shifting diversity where zooplankton diversity decreased and Cyanobacteria diversity increased. Second, we detected shifts in cyanotoxin phenology. After the zebra mussel invasion, microcystin increased in early summer and the duration of toxin production increased by over a month. Third, we observed shifts in heterotrophic bacteria phenology. The Bacteroidota phylum and members of the acI Nanopelagicales lineage were differentially more abundant. The proportion of the bacterial community that changed differed by season; spring and clearwater communities changed most following the spiny water flea invasion that lessened clearwater intensity, while summer communities changed least following the zebra mussel invasion despite the shifts in Cyanobacteria diversity and toxicity. A modeling framework identified the invasions as primary drivers of the observed phenological changes. These long-term invasion-mediated shifts in microbial phenology demonstrate the interconnectedness of microbes with the broader food web and their susceptibility to long-term environmental change.

Transcriptionally active nasopharyngeal commensals and opportunistic microbial dynamics define mild symptoms in the COVID 19 vaccination breakthroughs.

The development of COVID 19 vaccines as an effort to mitigate the outbreak, has saved millions of lives globally. However, vaccination breakthroughs have continuously challenged the vaccines' effectiveness and provided incentives to explore facets holding potential to alter vaccination-induced immunity and protection from subsequent infection, especially VOCs (Variants Of Concern). We explored the functional dynamics of nasopharyngeal transcriptionally active microbes (TAMs) between vaccination breakthroughs and unvaccinated SARS-CoV-2 infected individuals. Microbial taxonomic communities were differentially altered with skewed enrichment of bacterial class/genera of Firmicutes and Gammaproteobacteria with grossly reduced phylum Bacteroidetes in vaccination breakthrough individuals. The Bacillus genus was abundant in Firmicutes in vaccination breakthrough whereas Prevotella among Bacteroides dominated the unvaccinated. Also, Pseudomonas and Salmonella of Gammaproteobacteria were overrepresented in vaccination breakthrough, whilst unvaccinated showed presence of several genera, Achromobacter, Bordetella, Burkholderia, Neisseria, Hemophilus, Salmonella and Pseudomonas, belonging to Proteobacteria. At species level, the microbiota of vaccination breakthrough exhibited relatively higher abundance of unique commensals, in comparison to potential opportunistic microbes enrichment in unvaccinated patients' microbiota. Functional metabolic pathways like amino acid biosynthesis, sulphate assimilation, fatty acid and beta oxidation, associated with generation of SCFAs (short chain fatty acids), were enriched in vaccination breakthroughs. Majorly, metabolic pathways of LCFAs biosynthesis (long chain fatty acids; oleate, dodecenoate, palmitoleate, gondoate) were found associated with the unvaccinated. Our research highlights that vaccination decreases the microbial diversity in terms of depleting opportunistic pathogens and increasing the preponderance of commensals with respect to unvaccinated patients. Metabolic pathway analysis substantiates the shift in diversity to functionally modulate immune response generation, which may be related to mild clinical manifestations and faster recovery times during vaccination breakthroughs.

Alterations in gut microbiota and bile acids by proton-pump inhibitor use and possible mediating effects on elevated glucose levels and insulin resistance.

Several observational studies have suggested that proton-pump inhibitor (PPI) use might increase diabetes risk, but the mechanism remains unclear. This study aimed to investigate the effects of PPI use on gut microbiota and bile acids (BAs) profiles, and to explore whether these changes could mediate the association of PPIs use with fasting blood glucose (FBG) levels and insulin resistance (IR) in Chinese population. A cross-sectional study was conducted in Shenzhen, China, from April to August 2021, enrolled 200 eligible patients from the local hospital. Participants completed a questionnaire and provided blood and stool samples. Gut microbiome was measured by16S rRNA gene sequencing, and bile acids were quantified by UPLC-MS/MS. Insulin resistance (IR) was assessed using the Homeostasis Model Assessment 2 (HOMA2-IR). PPI use was positively associated with higher levels of FBG and HOMA2-IR after controlling for possible confounders. PPI users exhibited a decreased Firmicutes and an increase in Bacteroidetes phylum, alongside higher levels of glycoursodeoxycholic acid (GUDCA) and taurochenodeoxycholic acid (TCDCA). Higher abundances of Bacteroidetes and Fusobacterium as well as higher levels of TCDCA in PPI users were positively associated with elevated FBG or HOMA2-IR. Mediation analyses indicated that the elevated levels of FBG and HOMA2-IR with PPI use were partially mediated by the alterations in gut microbiota and specific BAs (i.e., Fusobacterium genera and TCDCA). Long-term PPI use may increase FBG and HOMA2-IR levels, and alterations in gut microbiota and BAs profiles may partially explain this association.

The role of morphine- and fentanyl-induced impairment of intestinal epithelial antibacterial activity in dysbiosis and its impact on the microbiota-gut-brain axis.

Recent evidence suggests that chronic exposure to opioid analgesics such as morphine disrupts the intestinal epithelial layer and causes intestinal dysbiosis. Depleting gut bacteria can preclude the development of tolerance to opioid-induced antinociception, suggesting an important role of the gut-brain axis in mediating opioid effects. The mechanism underlying opioid-induced dysbiosis, however, remains unclear. Host-produced antimicrobial peptides (AMPs) are critical for the integrity of the intestinal epithelial barrier as they prevent the pathogenesis of the enteric microbiota. Here, we report that chronic morphine or fentanyl exposure reduces the antimicrobial activity in the ileum, resulting in changes in the composition of bacteria. Fecal samples from morphine-treated mice had increased levels of Akkermansia muciniphila with a shift in the abundance ratio of Firmicutes and Bacteroidetes. Fecal microbial transplant (FMT) from morphine-naïve mice or oral supplementation with butyrate restored (a) the antimicrobial activity, (b) the expression of the antimicrobial peptide, Reg3γ, (c) prevented the increase in intestinal permeability and (d) prevented the development of antinociceptive tolerance in morphine-dependent mice. Improved epithelial barrier function with FMT or butyrate prevented the enrichment of the mucin-degrading A. muciniphila in morphine-dependent mice. These data implicate impairment of the antimicrobial activity of the intestinal epithelium as a mechanism by which opioids disrupt the microbiota-gut-brain axis.

Human gut bacteria contain acquired interbacterial defence systems.

The human gastrointestinal tract consists of a dense and diverse microbial community, the composition of which is intimately linked to health. Extrinsic factors such as diet and host immunity are insufficient to explain the constituents of this community, and direct interactions between co-resident microorganisms have been implicated as important drivers of microbiome composition. The genomes of bacteria derived from the gut microbiome contain several pathways that mediate contact-dependent interbacterial antagonism1-3. Many members of the Gram-negative order Bacteroidales encode the type VI secretion system (T6SS), which facilitates the delivery of toxic effector proteins into adjacent cells4,5. Here we report the occurrence of acquired interbacterial defence (AID) gene clusters in Bacteroidales species that reside within the human gut microbiome. These clusters encode arrays of immunity genes that protect against T6SS-mediated intra- and inter-species bacterial antagonism. Moreover, the clusters reside on mobile elements, and we show that their transfer is sufficient to confer resistance to toxins in vitro and in gnotobiotic mice. Finally, we identify and validate the protective capability of a recombinase-associated AID subtype (rAID-1) that is present broadly in Bacteroidales genomes. These rAID-1 gene clusters have a structure suggestive of active gene acquisition and include predicted immunity factors of toxins derived from diverse organisms. Our data suggest that neutralization of contact-dependent interbacterial antagonism by AID systems helps to shape human gut microbiome ecology.

SRS-FISH: A high-throughput platform linking microbiome metabolism to identity at the single-cell level.

One of the biggest challenges in microbiome research in environmental and medical samples is to better understand functional properties of microbial community members at a single-cell level. Single-cell isotope probing has become a key tool for this purpose, but the current detection methods for determination of isotope incorporation into single cells do not allow high-throughput analyses. Here, we report on the development of an imaging-based approach termed stimulated Raman scattering-two-photon fluorescence in situ hybridization (SRS-FISH) for high-throughput metabolism and identity analyses of microbial communities with single-cell resolution. SRS-FISH offers an imaging speed of 10 to 100 ms per cell, which is two to three orders of magnitude faster than achievable by state-of-the-art methods. Using this technique, we delineated metabolic responses of 30,000 individual cells to various mucosal sugars in the human gut microbiome via incorporation of deuterium from heavy water as an activity marker. Application of SRS-FISH to investigate the utilization of host-derived nutrients by two major human gut microbiome taxa revealed that response to mucosal sugars tends to be dominated by Bacteroidales, with an unexpected finding that Clostridia can outperform Bacteroidales at foraging fucose. With high sensitivity and speed, SRS-FISH will enable researchers to probe the fine-scale temporal, spatial, and individual activity patterns of microbial cells in complex communities with unprecedented detail.

A widely distributed phosphate-insensitive phosphatase presents a route for rapid organophosphorus remineralization in the biosphere.

The regeneration of bioavailable phosphate from immobilized organophosphorus represents a key process in the global phosphorus cycle and is facilitated by enzymes known as phosphatases. Most bacteria possess at least one of three phosphatases with broad substrate specificity, known as PhoA, PhoX, and PhoD, whose activity is optimal under alkaline conditions. The production and activity of these phosphatases is repressed by phosphate availability. Therefore, they are only fully functional when bacteria experience phosphorus-limiting growth conditions. Here, we reveal a previously overlooked phosphate-insensitive phosphatase, PafA, prevalent in Bacteroidetes, which is highly abundant in nature and represents a major route for the regeneration of environmental phosphate. Using the enzyme from Flavobacterium johnsoniae, we show that PafA is highly active toward phosphomonoesters, is fully functional in the presence of excess phosphate, and is essential for growth on phosphorylated carbohydrates as a sole carbon source. These distinct properties of PafA may expand the metabolic niche of Bacteroidetes by enabling the utilization of abundant organophosphorus substrates as C and P sources, providing a competitive advantage when inhabiting zones of high microbial activity and nutrient demand. PafA, which is constitutively synthesized by soil and marine flavobacteria, rapidly remineralizes phosphomonoesters releasing bioavailable phosphate that can be acquired by neighboring cells. The pafA gene is highly diverse in plant rhizospheres and is abundant in the global ocean, where it is expressed independently of phosphate availability. PafA therefore represents an important enzyme in the context of global biogeochemical cycling and has potential applications in sustainable agriculture.

A gut microbial peptide and molecular mimicry in the pathogenesis of type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of pancreatic ß-cells. One of the earliest aspects of this process is the development of autoantibodies and T cells directed at an epitope in the B-chain of insulin (insB:9-23). Analysis of microbial protein sequences with homology to the insB:9-23 sequence revealed 17 peptides showing >50% identity to insB:9-23. Of these 17 peptides, the hprt4-18 peptide, found in the normal human gut commensal Parabacteroides distasonis, activated both human T cell clones from T1D patients and T cell hybridomas from nonobese diabetic (NOD) mice specific to insB:9-23. Immunization of NOD mice with P. distasonis insB:9-23 peptide mimic or insB:9-23 peptide verified immune cross-reactivity. Colonization of female NOD mice with P. distasonis accelerated the development of T1D, increasing macrophages, dendritic cells, and destructive CD8+ T cells, while decreasing FoxP3+ regulatory T cells. Western blot analysis identified P. distasonis-reacting antibodies in sera of NOD mice colonized with P. distasonis and human T1D patients. Furthermore, adoptive transfer of splenocytes from P. distasonis-treated mice to NOD/SCID mice enhanced disease phenotype in the recipients. Finally, analysis of human children gut microbiome data from a longitudinal DIABIMMUNE study revealed that seroconversion rates (i.e., the proportion of individuals developing two or more autoantibodies) were consistently higher in children whose microbiome harbored sequences capable of producing the hprt4-18 peptide compared to individuals who did not harbor it. Taken together, these data demonstrate the potential role of a gut microbiota-derived insB:9-23-mimic peptide as a molecular trigger of T1D pathogenesis.

Characterization of rumen microbiome and immune genes expression of crossbred beef steers with divergent residual feed intake phenotypes.

We investigated whole blood and hepatic mRNA expressions of immune genes and rumen microbiome of crossbred beef steers with divergent residual feed intake phenotype to identify relevant biological processes underpinning feed efficiency in beef cattle. Low-RFI beef steers (n = 20; RFI = - 1.83 kg/d) and high-RFI beef steers (n = 20; RFI = + 2.12 kg/d) were identified from a group of 108 growing crossbred beef steers (average BW = 282 ± 30.4 kg) fed a high-forage total mixed ration after a 70-d performance testing period. At the end of the 70-d testing period, liver biopsies and blood samples were collected for total RNA extraction and cDNA synthesis. Rumen fluid samples were also collected for analysis of the rumen microbial community. The mRNA expression of 84 genes related to innate and adaptive immunity was analyzed using pathway-focused PCR-based arrays. Differentially expressed genes were determined using P-value ≤ 0.05 and fold change (FC) ≥ 1.5 (in whole blood) or ≥ 2.0 (in the liver). Gene ontology analysis of the differentially expressed genes revealed that pathways related to pattern recognition receptor activity, positive regulation of phagocytosis, positive regulation of vitamin metabolic process, vascular endothelial growth factor production, positive regulation of epithelial tube formation and T-helper cell differentiation were significantly enriched (FDR < 0.05) in low-RFI steers. In the rumen, the relative abundance of PeH15, Arthrobacter, Moryella, Weissella, and Muribaculaceae was enriched in low-RFI steers, while Methanobrevibacter, Bacteroidales_BS11_gut_group, Bacteroides and Clostridium_sensu_stricto_1 were reduced. In conclusion, our study found that low-RFI beef steers exhibit increased mRNA expression of genes related to immune cell functions in whole blood and liver tissues, specifically those involved in pathogen recognition and phagocytosis regulation. Additionally, these low-RFI steers showed differences in the relative abundance of some microbial taxa which may partially account for their improved feed efficiency compared to high-RFI steers.

Transcriptomic analysis of polysaccharide utilization loci reveals substrate preferences in ruminal generalists Segatella bryantii TF1-3 and Xylanibacter ruminicola KHP1.

Bacteria of the genera Xylanibacter and Segatella are among the most dominant groups in the rumen microbiota. They are characterized by the ability to utilize different hemicelluloses and pectin of plant cell-wall as well as plant energy storage polysaccharides. The degradation is possible with the use of cell envelope bound multiprotein apparatuses coded in polysaccharide utilization loci (PULs), which have been shown to be substrate specific. The knowledge of PUL presence in rumen Xylanibacter and Segatella based on bioinformatic analyses is already established and transcriptomic and genetic approaches confirmed predicted PULs for a limited number of substrates. In this study, we transcriptomically identified additional different PULs in Xylanibacter ruminicola KHP1 and Segatella bryantii TF1-3. We also identified substrate preferences and found that specific growth rate and extent of growth impacted the choice of substrates preferentially used for degradation. These preferred substrates were used by both strains simultaneously as judged by their PUL upregulation. Lastly, ß-glucan and xyloglucan were used by these strains in the absence of bioinformatically and transcriptomically identifiable PUL systems.

Comparing the gut microbiota of Sichuan golden monkeys across multiple captive and wild settings: roles of anthropogenic activities and host factors.

BACKGROUND: Captivity and artificial food provision are common conservation strategies for the endangered golden snub-nosed monkey (Rhinopithecus roxellana). Anthropogenic activities have been reported to impact the fitness of R. roxellana by altering their gut microbiota, a crucial indicator of animal health. Nevertheless, the degree of divergence in gut microbiota between different anthropogenically-disturbed (AD) R. roxellana and their counterparts in the wild has yet to be elucidated. Here, we conducted a comparative analysis of the gut microbiota across nine populations of R. roxellana spanning China, which included seven captive populations, one wild population, and another wild population subject to artificial food provision. RESULTS: Both captivity and food provision significantly altered the gut microbiota. AD populations exhibited common variations, such as increased Bacteroidetes and decreased Firmicutes (e.g., Ruminococcus), Actinobacteria (e.g., Parvibacter), Verrucomicrobia (e.g., Akkermansia), and Tenericutes. Additionally, a reduced Firmicutes/Bacteroidetes ratiosuggested diminished capacity for complex carbohydrate degradation in captive individuals. The results of microbial functional prediction suggested that AD populations displayed heightened microbial genes linked to vitamin and amino acid metabolism, alongside decreased genes associated antibiotics biosynthesis (e.g., penicillin, cephalosporin, macrolides, and clavulanic acid) and secondary metabolite degradation (e.g., naphthalene and atrazine). These microbial alterations implied potential disparities in the health status between AD and wild individuals. AD populations exhibited varying degrees of microbial changes compared to the wild group, implying that the extent of these variations might serve as a metric for assessing the health status of AD populations. Furthermore, utilizing the individual information of captive individuals, we identified associations between variations in the gut microbiota of R. roxellana and host age, as well as pedigree. Older individuals exhibited higher microbial diversity, while a closer genetic relatedness reflected a more similar gut microbiota. CONCLUSIONS: Our aim was to assess how anthropogenic activities and host factors influence the gut microbiota of R. roxellana. Anthropogenic activities led to consistent changes in gut microbial diversity and function, while host age and genetic relatedness contributed to interindividual variations in the gut microbiota. These findings may contribute to the establishment of health assessment standards and the optimization of breeding conditions for captive R. roxellana populations.