Animal Microbiome

Diet influences the composition, diversity, and functional capacity of the cattle gut microbiome. However, the extent to which feeding practices affect the microbial community and resistome under real-world conditions remains poorly understood, particularly in low- and middle-income settings. Here, we applied metagenomics to fecal samples from Bangladeshi cattle fed either a natural or a mixed diet to examine differences in microbial composition, functional potential, and resistome associated with feed type. Natural-fed cattle harbored higher microbial diversity and distinct bacterial phyla, including Bacteroidota, Campylobacteriota, and Mycoplasmatota. Acinetobacter, Aliarcobacter, Comamonas, Dysosmobacter, and Sharpea were enriched in natural-fed cattle, whereas Anaerotignum, Aristaeella, Oscillibacter, and Clostridium were more abundant in the mixed-fed group. Notably, the emerging zoonotic genus Aliarcobacter was detected in the natural-fed cohort. Alpha diversity analysis showed higher richness and evenness in natural-fed cattle, and a clear separation between dietary groups in beta diversity analysis (PERMANOVA, p = 0.01). Differential analysis identified Oscillibacter ruminantium as a biomarker of natural feeding, while Succinivibrio faecicola and Anaerovibrio slackiae for mixed feeding. Resistome profiles demonstrated clear differences. Mixed-fed cattle showed a consistent enrichment of tetracycline resistance genes, whereas the natural-fed group displayed a more variable resistome. Functional analysis suggested diet-associated differences in metabolic potential, with glutathione metabolism enriched in natural-fed cattle (p<0.05) and bile secretion and fatty acid metabolism moderately enriched in the mixed-fed group. These findings indicate that feeding practices are associated with differences in rumen microbial communities and resistome profiles in Bangladeshi cattle, providing baseline insights into microbiome-resistome relationships under field conditions.

The evolutionary divergence between Henophidia (non-venomous) and Caenophidia (venomous) snakes has produced distinct cranial morphologies, digestive strategies, and presence of specialised venom systems in Caenophidia, yet the extent to which these long-standing diverging trajectories have shaped cloacal microbiota assembly remains poorly understood. We characterised cloacal microbiota in 70 captive snakes (52 Caenophidia, 18 Henophidia) by 16S rRNA amplicon sequencing. Beta diversity was tested by PERMANOVA, differential abundance by ANCOM-BC2, community types by Dirichlet Multinomial Mixture modelling (DMM), and microbial interactions by SparCC co-occurrence networks. Predicted functional potential (PICRUSt2) was analysed by ALDEx2 differential abundance testing and elastic net feature selection. Henophidia exhibited significantly higher bacterial richness and greater compositional variability than Caenophidia. Community composition showed clade-associated differences (PERMANOVA) and partitioned into two distinct DMM community types. The Henophidia network was 11.9-fold denser and more modular, with Burkholderiaceae as a keystone hub, whereas the Caenophidia network was sparse. Henophidia showed predicted enrichment in C1 metabolic pathways (ethylmalonyl-CoA, formaldehyde assimilation I, glycine betaine degradation I, methylaspartate cycle), aromatic compound catabolism, and nitrogen recycling, whilst Caenophidia showed enrichment in allantoin and glucuronate degradation. This multi-method analysis suggests Burkholderiaceae as a candidate keystone taxon in Henophidia and indicates that phylogenetic clade is a major contributor to cloacal microbiota structure. The lower richness in Caenophidia raises a testable hypothesis that broad-spectrum antimicrobial activity of their venom components may selectively filter susceptible microbial lineages, motivating future shotgun metagenomic studies in wild populations of snakes.

The fish skin microbiome serves as a protective barrier, influencing host health and facilitating interactions between the host and its environment. While several studies have characterised the composition and roles of the fish skin microbiome, there remains a paucity of data on how environmental variation influences these microbes in natural populations. Here, we used 16S rRNA gene sequencing to characterise the skin microbiome of wild three-spined stickleback populations and examine how environmental factors influence microbial diversity and community composition across 17 freshwater lochs on the island of North Uist, Scotland. Analysis of 239 samples revealed a set of dominant bacterial genera commonly associated with other fish species, including Janthinobacterium, Pseudomonas, Acinetobacter, and Psychrobacter, that constituted a core skin microbiota across lochs. Microbiome composition was primarily shaped by environmental variables, particularly habitat, water pH, conductivity, and metal concentrations, with pH emerging as a key driver of community structure. Host sex also influenced microbiome variation, with several taxa differing in relative abundance between males and females. Alpha-diversity was higher among stickleback fish from lochs with a neutral pH compared with those from alkaline and acidic environments. Differential abundance analyses identified 27 and 24 amplicon sequence variants (ASVs), respectfully, associated with variations in pH and host sex, including members of Psychrobacter, Sphingobacterium, Carnobacterium, Chryseobacterium, and Arthrobacter, highlighting the combined influence of environmental and host factors on microbiome composition in wild fish populations in freshwater environments.

1.Amplicon-based profiling of airway microbiota is increasingly used to assess respiratory health in mammals, yet baseline data for free-ranging baleen whales remain scarce. We characterised the exhaled-breath ("blow") microbiota of rorqual whales in the Gulf of St. Lawrence (Canada) and examined associations with individual health indicators. Blow samples were collected opportunistically from six whales (two blue, two fin and two humpback), with seawater and air controls. The V4 region of the 16S rRNA gene was sequenced on an Illumina MiSeq platform and processed in R (v4.5) using the DADA2 pipeline for quality filtering, denoising and amplicon sequence variant (ASV) inference. Alpha diversity varied among individuals (Shannon = 2.72 - 4.33) and beta-diversity analyses revealed a significant effect of environment (whale blow vs. seawater) on community composition (PERMANOVA: R2 = 0.140, F = 1.31, p = 0.030). The relative abundance of pathobionts (22.8-48.8%) was negatively correlated with alpha diversity (Spearman {rho} = -0.88 to -0.94, p < 0.05), while higher diversity correlated positively with good skin condition ({rho} = 0.84, p = 0.03). These findings provide the first baseline description of rorqual respiratory microbiota in the Gulf of St. Lawrence and support blow microbiome metrics as non-invasive health biomarkers.

Sarbecoviruses, a subgenus of Betacoronavirus, display both respiratory and gastrointestinal tropism, suggesting potential interactions with host gut microbial communities. However, ecological signatures of infection in wild bats remain poorly understood. We investigated associations between Sarbecovirus infection status, gut microbiome structure, and diet composition in Rhinolophus shameli roosting in northeastern Cambodia. Fecal samples collected across dry and wet seasons (2023-2024) were subject to full-length 16S rRNA gene sequencing and arthropod DNA metabarcoding. Sarbecovirus-positive bats exhibited stable alpha diversity but consistent shifts in gut community composition and increased interindividual variability consistent with the Anna Karenina Principle, suggesting infection-associated destabilization of community assembly rather than diversity erosion. Infection status was associated with enrichment of Shigella and Escherichia species, taxa linked to inflammatory or epithelial stress states in bats. In contrast, dietary composition showed no strong global structuring by infection status and weak coupling with bacterial community structure, suggesting that trophic ecology is unlikely to be the main driver of the infection-associated microbiome signal. Although causal directionality cannot be inferred, our results reveal measurable and consistent microbiome restructuring associated with Sarbecovirus detection in a natural reservoir host and highlight the potential of microbiome profiling for monitoring wildlife disease processes.

Germ-free (GF) animal models are indispensable for dissecting host-microbiota interactions and their roles in health and disease. The small teleost fish medaka (Oryzias latipes) provides unique advantages for establishing GF models across developmental stages, yet the functions of its intestinal microbiota and metabolites remain poorly characterized. Here, we developed both early-life and chronic GF medaka models to systematically characterize host biology in the absence of microbiota and evaluate the contribution of gut-derived metabolites to growth and immune development. Using a refined sterile feeding and verification protocol, we successfully maintained GF medaka for up to 57 days post-fertilization (dpf). As anticipated, GF fish displayed developmental delays, impaired organogenesis, reduced immune competence, and metabolic dysregulation. Supplementation with sterile gut-derived metabolites partially alleviated these deficits, as evidenced by enhanced locomotor activity and immune responses. Mechanistically, recovery was associated with improved ribosome biogenesis, tricarboxylic acid cycle activity, and histidine and pyruvate metabolism, suggesting enhanced protein synthesis and immune maturation. However, metabolite supplementation also elevated oxidative stress and inflammatory responses and failed to fully restore long-term survival or organ development. Our findings support the use of GF medaka as a versatile platform for investigating microbiota-host interactions across life stages. By integrating metabolite interventions, this model provides critical insights into the functional roles of gut microbiota and offers a valuable tool for advancing microbiome research in health and disease.

The female reproductive tract harbors complex microbial communities that may influence reproductive success. In previous work using 16S rRNA gene sequencing, we identified bacterial taxa in the vagina and uterus of beef cattle associated with pregnancy outcomes, but taxonomic resolution and functional inference was limited. Here we used shotgun metagenomic sequencing to characterize the taxonomic composition, functional potential, and antimicrobial resistome of vaginal and uterine microbiomes at the time of artificial insemination (AI) in cows that subsequently became pregnant or remained open. Vaginal (pregnant n = 54; open n = 7) and uterine (pregnant, n = 41; open, n = 9) samples were collected prior to AI. Microbial community structure did not differ between pregnancy outcome groups in either anatomical site (PERMANOVA; P > 0.05). However, cows that remained open showed significantly greater species-level richness and diversity in the vaginal microbiome (P < 0.05). No diversity differences were observed in the uterine microbiome. In contrast, significant differences were detected between anatomical sites, with distinct dominant taxa and functional profiles. Vaginal microbiomes were enriched in pathways related to genetic information processing, whereas uterine microbiomes exhibited greater representation of metabolic pathways. A total of 105 ARGs spanning 11 antimicrobial classes were identified, with tetracycline resistance genes [tet(Q), tet(W), and tet(M)] predominating, and blaTEM-116 more abundant in the uterine microbiome. Overall, while vaginal and uterine microbiomes were compositionally and functionally distinct, no robust pregnancy-associated taxonomic or functional signatures were detected, likely reflecting limited statistical power and challenges inherent to low-biomass metagenomic datasets. IMPORTANCEUnderstanding the role of the reproductive tract microbiome in fertility could improve reproductive efficiency in cattle. We used shotgun metagenomic sequencing to characterize the taxonomic composition, functional potential, and antimicrobial resistome of vaginal and uterine microbiomes at the time of artificial insemination in cows that subsequently became pregnant or remained open. Using paired samples from the same animals, we directly compared microbial communities between the upper and lower reproductive tract to identify shared and site-specific features. Although no distinct microbial signatures associated with pregnancy outcomes were detected, this may reflect limited statistical power and low microbial biomass inherent to these samples. Despite these challenges, our study provides high-resolution insights into the composition, functional potential, and resistome of bovine reproductive microbiomes and highlights important technical considerations for studying low-biomass microbial ecosystems.

It is well established that the microbiome can have major effects on animal biology. Here we have investigated the composition of the bacterial and eukaryotic gut microbiome of wild mice from three sample sites and sought to understand what affects its composition. We find that the bacterial and eukaryotic microbiome differs among mice from different sites. Among mouse traits, we found that only gut inflammation and the concentration of faecal immunoglobulin A affected the microbiome diversity. However, the microbiome diversity was more commonly affected by the microbiome composition itself, both within-bacterial and within-eukaryotic, but also by cross bacterial-eukaryotic effects. We found that most hosts produce IgA that binds some of their gut bacteria, though mice are largely idiosyncratic in which taxa they bind with IgA, with a few taxa commonly IgA-bound. The eukaryotic microbiome was dominated by fungal taxa, and included Eimeria infection that was particularly common at one of the sites. At the high Eimeria prevalence site, mice had comparatively marked caecal inflammation and significantly greater IgA responses. Our results emphasise the substantial among-individual mouse differences in gut microbiome composition, gut physiology and immunology, and the biological significance of the bacterial-eukaryotic effects that we suggest requires further study.

Spotty Liver Disease (SLD) is an acute bacterial infection of layer chickens in production, caused by Campylobacter hepaticus, and occurs most frequently in barn-housed and free-range systems. The disease is characterized by a sharp decline in egg production and increased mortality. The hallmark pathological feature is 1-2 mm white to grey necrotic foci distributed across the liver surface. Despite its growing economic impact on commercial poultry, the molecular mechanisms underlying host responses to C. hepaticus infection remain poorly understood. To address this gap, we performed a comprehensive transcriptome analysis of liver tissue from chickens naturally infected with SLD compared to uninfected controls. High-throughput transcriptome sequencing, yielding 9,277 differentially expressed genes (DEG), of which 3,063 were upregulated and 6,214 were downregulated. Functional pathway enrichment analysis revealed significant alterations in immune and metabolic processes associated with SLD pathophysiology. Infected chickens exhibited significant activation of immune response pathways, particularly cytokine-cytokine receptor interactions involving interleukins IL-22, IL-21, and IL-6, along with enhanced cell signaling, and cell adhesion. Among the individual genes, C1QTNF1 and the adhesion molecule gene ADGRD1 were notably overexpressed, indicating enhanced inflammatory activity. In contrast, core hepatic metabolic functions were profoundly reduced, as evidenced by downregulation of oxidative phosphorylation, fatty acid metabolism, iron ion binding, and heme binding pathways. A marked increase in serum amyloid A gene (SAA) expression further confirmed robust acute-phase responses and compromised liver function during infection. Together, these findings demonstrate a complex interplay between inflammatory activation and metabolic dysregulation during SLD. The strong upregulation of acute-phase proteins and pro-inflammatory cytokines demonstrates the hosts vigorous attempt to combat bacterial infection, whereas the concurrent suppression of essential metabolic pathways reflects the pathological consequences of SLD. This study provides a transcriptomic characterization of host responses to C. hepaticus infection, offering insights into SLD pathogenesis and potential avenues for targeted intervention.

Animal-associated bacteria (microbiomes) can have important effects on host phenotypes and fitness. Microbiomes can also vary across individuals in ways that depend on host genotype and environment. Temperature is an especially important environmental factor that can affect the microbiome in a way that depends on host genotype and affects organismal fitness. Thermal stress, in particular, can have dramatic effects on animal microbiomes, including dysbiosis and immune dysregulation. However, most previous work on extreme temperature effects has focused on heat stress. To investigate how low temperatures affect the microbiome of a warm-adapted animal, we characterized the bacterial communities associated with house fly (Musca domestica) males raised at cool (18{degrees}C) and warm (29{degrees}C) temperatures. We sampled two distinct genotypes in these experimental flies, each of which is associated with a particular thermal environment (warm or cool). We contrasted our experimental results with the microbiomes we characterized in wild house flies from two collection sites with different large animals present. We found that temperature has a much stronger effect on the house fly microbiome than the host genotype in our experimental flies. Consistent with the strong environmental effects in our experiment, we found that wild house fly microbiomes differed between the two collection sites. Despite these environmental effects on the house fly microbiome, we did not detect evidence for dysbiosis associated with either cool or warm temperatures. We therefore conclude that the environment has more of an effect on the house fly microbiome than host genotype, but dysbiosis does not occur within the temperature range we considered.

Centrohelid heliozoans are a monophyletic group of free-living, ubiquitous, predatory protists widely distributed in aquatic and soil ecosystems. Centrohelids are known as cytotrophic protists that feed on bacteria, algae, and small unicellular eukaryotes. While algal and chloroplast symbioses have been documented in this group, their bacterial associations remain largely unexplored. In this study, we characterize the bacterial communities associated with centrohelids isolated from freshwater habitats using full-length 16S rRNA PacBio sequencing. Amplicon sequencing revealed 5 phyla, 6 classes, and 58 genera in the bacterial communities associated with seven centrohelid isolates. Alphaproteobacteria, Bacteroidia, and Gammaproteobacteria were the most abundant classes, while Arcicella, Sphingobium, Pseudomonas, Sphingomonas, Azospirillum, Shinella, Flavobacterium, Variovorax, and Rhodococcus were the most abundant genera. Notably, Arcicella, Variovorax, Sphingobium, and Pseudomonas constituted the core microbiome. Unexpectedly, we detected bacteria known as opportunistic pathogens, providing the first evidence that centrohelids may serve as environmental reservoirs for bacteria with pathogenic potential (e.g., Acidovorax, Acinetobacter, Anaerococcus, Bosea, Corynebacterium, Escherichia, Moraxella, Mycobacterium, Prevotella, Pseudomonas, Ralstonia, and Sphingomonas). In addition, this study provides the first evidence of Rickettsiaceae associations with centrohelids. IMPORTANCEThis study reveals that centrohelid heliozoans, ubiquitous microbial predators, harbor diverse and host-specific bacterial communities. Critically, we show they can serve as environmental reservoirs for bacteria with pathogenic potential, a role previously overlooked outside of model protist groups. These findings expand our understanding of pathogen ecology, suggesting that a wider range of protists may contribute to the persistence and dispersal of opportunistic pathogens in aquatic ecosystems.

Microbial communities are essential for host health and ecosystem stability. However, whether host identity or shared foraging resources shapes microbiome structure among co-occurring species remains poorly understood. We studied bacterial and fungal communities of four Indian honeybee species in a mustard monoculture resource condition, integrating behavioural observation-based pollinator data with microbial co-occurrence networks derived from metabarcoding. Microbiome composition was linked to host identity rather than foraging behaviour, bee abundance, or landscape use. While core bacterial taxa were shared, relationships among bacterial cobionts, unlike those among fungal genera, remained species-specific. Microbial diversity, along with community structure and function, influenced network stability, with a highly modular microbial network of Apis cerana exhibiting more resilience to simulated perturbations. In summary, host-specific filtering shaped the microbiome more than resource homogenisation, with closely related species facing unique risks of microbial collapse, with broader implications for vulnerability to microbiome imbalance, environmental stress, and emerging infections.

Most gut microbiome studies have focused on bacteria, leaving a knowledge gap regarding gut associated fungi. We assessed fungal diversity in the gastrointestinal tract of the reptilian order Testudines (turtles and tortoises) using samples from 6 families, 19 genera, and 27 species. A highly diverse community affiliated with 17 phyla and 157 orders was encountered, with four phyla (Neocallimastigomycota, Chytridiomycota, Ascomycota, and Basidiomycota) representing 89.13% of the community. Neocallimastigomycota was identified in host families Testudinidae (land tortoises), Chelidae, Chelydridae, Emydidae, Geoemydidae, and Kinosternidae, with higher relative abundances in Testudinidae (40.18{+/-}37.97%) compared to all other families combined (2.71{+/-}4.04%). Neocallimastigomycota sequences were mostly affiliated with orders Testudinimycetales in the host family Testudinidae and Neocallimastigales in other host families. Chytridiomycota was identified in all host families, but was more ubiquitous and abundant in Kinosternidiae (45.17{+/-}34.12%), and exhibited a high level of variability across samples. Dikarya communities were highly diverse, with 108 orders identified, and, similar to Chytridiomoycota, exhibited a highly stochastic distribution pattern. Representatives of multiple yet-uncultured phyla (Candidatus "Algovoracomycota", "Sedimentomastigomycota", "Tartumycota" and "Cantoromastigomycota") were identified, as well as eight novel orders in Chytridiomycota and Rozellomycota. Deterministic selection shaped community assembly in the host family Testudinidae, while the process was more stochastic in other host families. Distinct community structure was driven by differences in abundance and identity of the Neocallimastigomycota when comparing Testudinidae to. Our results describe a diverse and dynamic fungal community, shaped by the co-occurrence of autochthonous (resident) and transient (allochthonous) members of the gut microbiome. ImportanceFungi are known to inhabit the gastrointestinal tract (GIT) of humans and mammals. However, information on the fungal community in the GIT of reptiles is relatively sparse. We investigated the diversity and community structure of fungi in the reptilian order Testudines. We conducted a culture-independent diversity survey on fecal samples obtained from 27 different host species. We identify representatives of 17 fungal phyla. As well, we demonstrate that the anaerobic gut fungi (phylum Neocallimastigomycota) are not restricted to the family Testudinidae (land tortoises) as previously suggested, but could successfully colonize and inhabit all other testudines families, including those exhibiting a predominantly omnivorous or carnivorous lifestyles. In addition, we expand on the known fungal diversity by identifying additional representatives of multiple recently described yet-uncultured phyla, and describe multiple novel orders and classes within existing phyla. Collectively, this effort adds to the growing body of knowledge of mycobiomes in underexplored animal hosts.

Urbanization can result in shifts in abiotic and biotic factors, including temperature, pollution, habitat type, pathogens, and diet, among others. These shifts can, in turn, shape the ecological and evolutionary trajectory of urban wildlife. The gut microbiota has the potential to mediate host-environment interactions, especially in the context of diet and disease, and thus may be a useful lens for understanding the impacts of urbanization. House mice (Mus musculus domesticus) are a cosmopolitan human commensal with a wealth of genomic and metagenomic resources. Here, we investigate patterns of variation in diet and gut microbial diversity, community composition, and function using a paired urban-rural sampling design in house mice from three metro regions in the eastern United States. First, using stable isotope analysis, we found that habitat--urban versus rural--was a major driver of variation in {delta}15N, suggesting a diet richer in animal proteins in cities. Next, using short-read sequencing of the 16S rRNA gene, we found that urban mice have lower gut microbial taxonomic diversity than their rural counterparts. We also found that community composition varied among urban and rural habitats, with differences largely reflecting shifts among closely related taxa. In particular, Prevotellaceae, a family known to be responsive to dietary quality, was differentially abundant, with lower abundance in urban habitats. Finally, we found differentiation in a few predicted microbial functions across habitat, primarily related to metabolism. Together, data across three independent sampling regions provide strong evidence that urbanization has the potential to shape the diet and the microbiome of house mice.

Salmonella spp. represents a leading cause of foodborne disease globally. Wild aquatic birds inhabiting ecosystems impacted by human activities may serve as reservoirs and dispersers of Salmonella and antimicrobial resistance genes (ARGs), posing significant public health risks. This study evaluated the prevalence, serovars, resistance genes, and genomic relationships of Salmonella in fecal samples from wild aquatic birds across three high-Andean lakes in Ecuador. Of 134 samples collected from 10 species, five (3.73%) tested positive, all from Yahuarcocha Lake, isolated from Fulica ardesiaca and Phalacrocorax brasilianus. Two serovars were identified: Salmonella Infantis (ST32, n=4) and Salmonella Newport (ST45, n=1). Three S. Infantis isolates exhibited multidrug resistance (MDR), mediated by a pESI-like plasmid carrying resistance genes against beta-lactams, aminoglycosides, tetracyclines, sulfonamides, trimethoprim, fosfomycin, and chloramphenicol. SNP-based phylogenetic analysis revealed low genetic divergence ([&le;]10 SNPs) between wildlife and poultry-associated isolates, indicating a shared transmission network. These findings support a likely spillover from poultry production systems into wild bird populations, and highlight the role of wild aquatic birds as ecological sentinels and potential disseminators of MDR Salmonella across interconnected human, animal, and environmental systems. These results underscore the need to incorporate human, animal, and environmental health factors within a One Health framework.

Bilophila wadsworthia is a gut pathobiont implicated in dysbiosis-driven inflammation, yet its pathogenic mechanisms remain poorly investigated. Here, we evaluated the suitability of Galleria mellonella larvae as an in vivo model to study B. wadsworthia infection. Two infection routes were compared: oral inoculation to mimic gastrointestinal colonization and hemolymph injection to model systemic infection. Oral challenge had minimal impact on larval health, whereas hemolymph injection caused marked morbidity, including reduced mobility, impaired cocoon formation, and progressive melanization, indicating that access to the circulatory system is required for overt disease. Infection required live bacteria, with B. wadsworthia capable of intracellular replication within hemocytes, leading to transient depletion of circulating immune cells followed by compensatory hemocyte proliferation. These findings reveal tight coupling between bacterial proliferation and host immune dynamics. Comparison with other sulfidogenic bacteria suggests that Bilophila pathogenicity is likely to involve host-specific interactions. Overall, our results establish G. mellonella as a practical and ethically favorable model to investigate B. wadsworthia virulence, host-pathogen interactions, and mechanisms relevant to gut-associated infection.

Microbial communities are critical to the functioning of ecosystems and shape the ecology and evolution of host organisms. However, we have a limited understanding of how host-associated and free-living microbes differ in their structure and biogeography. Here, we test whether host-associated (fish gut) and free-living (lake bacterioplankton) microbes exhibit different metacommunity structure, spatial turnover, and consistency with neutral expectations using two independent lake systems. We characterized microbial communities in lake water (Vancouver Island and Sierra Nevada) and guts in two fish species (stickleback and brook trout) using 16S amplicon sequencing. We compared alpha and beta diversity within lakes, quantified spatial turnover (distance-decay), and tested for departure from neutral abundance-occurrence expectations between bacterioplankton and fish gut microbiomes. Fish microbiomes had lower alpha diversity compared to bacterioplankton, but higher beta diversity within lakes. Bacterioplankton were more similar across lakes yet showed stronger patterns of spatial turnover with distance than fish gut microbiomes. A neutral model explained a substantial proportion of abundance-occurrence relationships in bacterioplankton communities but performed poorly for fish-associated microbes. Our study indicates that host-associated and free-living microbes have disparate patterns of metacommunity structure and spatial turnover consistent with differences in the strength of neutral ecological processes. Fish microbiomes were less diverse at the local scale but more variable across space and time than bacterioplankton communities, suggestive of potentially strong local selection and/or reduced microbial exchange among hosts compared to environmental communities. Importantly, we observed highly consistent patterns across both lake systems despite differences in host species, sampling design, and region, demonstrating that differences in the distribution of host and environmental microbes are potentially widespread. This study demonstrates how host association fundamentally alters the diversity and spatial distribution of microbes, emphasizing the need to incorporate hosts into broader frameworks of microbial biogeography.

Holometabolous polyphagous insects undergo complete metamorphosis and exploit multiple host plants, exposing them to highly variable ecological conditions across both life stages and host plants. Whether these species, like specialist ones, harbour a stable core microbiota, or whether life stages or host plants act as the primary drivers of microbiota assembly remain open questions. Here, we characterized the fungal and bacterial communities associated with Drosophila suzukii across life stages and host fruits using 16S and ITS metabarcoding. We tested the relative influence of life stage and host fruit on microbiota composition, using community and network-based analyses. We first identified that host fruit significantly structured fungal communities, but not bacterial ones. Yeast communities were rather fruit-specific: Hanseniaspora and Pichia mostly associated with cherries and strawberries, contrary to Metschnikowia with blackberries. In contrast, bacteria and filamentous fungi were shared across fruits, constituting for fruits a core microbiota dominated by Gluconobacter cerinus, Tatumella and Cladosporium. Second, we found that both bacterial and fungal D. suzukii communities were structured by life stage, and that fungal, but not bacterial communities, were also structured by host fruits. D. suzukii individuals harboured a core bacteria composed of G. cerinus and a niche-specific microbiota composed of yeasts: Hanseniaspora typical in individuals related to cherry and strawberry, and Metschnikowia to blackberry. Components of both core and niche-specific microbiota were most likely horizontally acquired by D. suzukii from host fruits. Taken together our results underline the importance of meta-community approaches to investigate tripartite interactions among insects, host plants and microbiota. IMPORTANCEThe role of gut microbiota in mediating interactions between phytophagous insects and their host plants has been well illustrated in specialist species. However, it has been less comprehensively studied in polyphagous species, which infest multiple host plants, and across life stages for holometabolous species experiencing separate ecological niches through development. We tested the existence of a core, a niche-specific and a stage-specific microbiota in a polyphagous holometabolous species, D. suzukii. We examined both fungal and bacterial communities in larvae, pupae and emerging flies infesting three host fruits. Our results showed first that the assembly of bacteria, filamentous fungi and yeasts on fruits is driven by different ecological processes. Second, that D. suzukii harbours a core bacterial microbiota, a niche-specific microbiota constituted by yeasts and no stage-specific microbiota. Our study emphasizes the importance of considering jointly the assembly of host plant and polyphagous insect microbial communities to better understand the ecology and evolution of insect-microbe interactions.

Illegal wildlife trade and habitat degradation displace thousands of animals annually in Southeast Asia, with many confiscated primates housed in rehabilitation centers that increasingly function as long-term holding environments. In slow lorises, dental clipping associated with the pet trade may generate persistent disruption along the oral-gut axis, potentially undermining physiological readiness for release in ways not captured by conventional screening. Here, we evaluated whether microbiome structure provides an integrative marker of release readiness in rehabilitating Javan slow lorises (Nycticebus javanicus). From June to October 2024, we collected fecal (n = 26) and saliva (n = 18) samples from 19 adults housed at YIARI, including 10 release candidates and 9 non-candidates classified primarily based on tooth loss, medical history, and possibility of release. Bacterial communities were characterized using 16S rRNA (V3-V4) amplicon sequencing, with alpha and beta diversity, taxonomic enrichment (LEfSe), and predicted functional profiles (PICRUSt2) assessed. Microbiome composition was strongly compartmentalized by body site, with higher alpha diversity in the gut. Release candidacy was associated with modest gut compositional differences, whereas oral microbiomes showed pronounced divergence between candidates and non-candidates. Non-candidates were enriched in dysbiosis-associated taxa and degradation-oriented functional pathways, while candidates showed enrichment of biosynthetic and central energy metabolism pathways. Gut microbiome structure was stable across pre-release and soft-release phases. These findings indicate that oral and gut microbiomes represent distinct physiological niches and that persistent oral microbiome alteration is a sensitive marker of long-term dental perturbation. Integrating microbiome-informed metrics may improve multidimensional assessment of release readiness.

Foodborne non-typhoidal Salmonella remains a major public health concern, yet routine surveillance recovers large numbers of isolates from food that are not associated with human illness. Studies have shown foodborne isolates can be genetically linked to clinical cases, highlighting a critical challenge for risk assessment and outbreak prioritisation. This study aimed to determine whether genomic markers can distinguish foodborne Salmonella strains with an increased likelihood of causing infection. Whole-genome sequencing data from over 900 Salmonella isolates recovered from food and the environment through UK Health Security Agency surveillance were analysed using hierarchical clustering to define genetically related groups. These clusters were expanded using the global EnteroBase database to provide broader epidemiological context. Genome-wide association analyses identified genetic markers associated with clusters containing clinical isolates, including phage-associated regions. A highly conserved 7 kb marker identified in S. Agona demonstrated strong predictive performance at a global scale, with high sensitivity and specificity for infection-associated lineages and strict serovar restriction. Comparative genomic analysis revealed that all markers localised to a shared chromosomal hotspot corresponding to a prophage integration site. The 7 kb risk-associated marker formed part of a larger prophage closely related to the well-characterised S. Typhimurium Fels-2 phage, which encodes a DNA invertase linked to phase variation, a mechanism known to promote phenotypic heterogeneity and host adaptation. As these S. Agona isolates are monophasic, our findings indicate that our genome-wide association approach has rediscovered this DNA invertase known to contribute to infection risk but in a different serovar via an alternative regulatory mechanism. Overall, this work demonstrates the potential to move beyond treating all foodborne Salmonella isolates as equivalent hazards, towards a genomics-informed framework for risk stratification. This approach provides a foundation for improved risk-based decision-making, enhance outbreak investigations and enable earlier prioritisation of public health responses during Salmonella surveillance and control. Author summaryFoodborne Salmonella infections remain a major public health concern, but not all strains pose the same risk to human health. Here we investigated whether genetic differences could explain why some foodborne strains are more likely to cause human infection. We analysed over 900 genomes from food and environmental sources, grouping closely related strains before placing them in a global context using EnteroBase. By combining pangenome and genome-wide association analyses, we identified distinct lineages within several serovars that differed in their association with human cases. In Salmonella Agona, all clinical isolates belonged to a single lineage carrying a highly conserved 7 kb marker that was absent from low-risk strains. This marker demonstrated strong sensitivity and specificity across global datasets and was located within a prophage closely related to the well-characterised Fels-2 phage. This region encodes a DNA invertase previously linked to phase variation, a mechanism that promotes bacterial adaptability. Our findings indicate that infection risk can be structured at the lineage level and influenced by mobile genomic elements, particularly prophages, that enhance environmental persistence and host adaptation. This work advances genomic surveillance from retrospective linkage towards mechanistic and predictive risk assessment, with direct relevance for supporting risk-based decision-making during outbreak investigations.