Animal Microbiome

Advances in metagenomic sequencing have transformed our understanding of host-associated microbiota, revealing critical roles in immune regulation, nutrient metabolism, and gastrointestinal (GI) homeostasis. However, the gut microbiome of large non-model species, particularly elephants, remains poorly characterized. Captivity introduces dietary, environmental, and management-related perturbations that may disrupt microbial balance and contribute to GI dysfunction. Here, we performed a longitudinal analysis of the fecal microbiome in five captive Asian elephants (Elephas maximus) at the Oregon Zoo exhibiting chronic fecal abnormalities, including mucus and inconsistent stool formation. Over 14 weeks, weekly fecal samples were collected and compared with samples from clinically normal elephants housed at three other zoological institutions. Using 16S rRNA gene sequencing, we identified marked differences in microbial community composition between affected and control elephants. Dysbiosis in Oregon Zoo elephants was characterized by enrichment of Akkermansia muciniphila and multiple members of the order Clostridiales, including taxa previously associated with gastrointestinal disorders. Administration of a commercially available probiotic formulation was associated with transient improvement in fecal characteristics and pronounced shifts in microbial composition, including a significant post-treatment reduction in overall microbial diversity and decreased abundance of several taxa linked to GI abnormalities. Notably, probiotic strains themselves were not detected, suggesting indirect or short-lived functional effects rather than durable colonization. Together, these findings provide one of the first longitudinal characterizations of gut microbiome dysbiosis in captive Asian elephants and identify candidate microbial contributors to chronic GI dysfunction in captivity, with implications for husbandry, dietary management, and microbiome-informed interventions in megafauna. Additionally, our study underscores the potential, although limited and likely indirect, benefit of probiotics when treating GI disorders in monograstric megavertebrates.

The gut microbiome is increasingly recognized as a pivotal modulator of animal behaviour, yet its influence on wild fauna remains largely unexplored. We investigated the correlational relationship between gut microbiota, behavioural phenotypes, and management practices in 26 captive endangered Iberian lynxs (Lynx pardinus) maintained within the ex-situ Iberian breeding program facilities, in two geographically distant stations in SW Spain. Behavioural observations were intensively recorded over two years, and three personality profiles emerged, i.e., (i) anomalous (with the highest frequencies for stereotypies), and (ii) sedentary and (iii) active (with the highest frequencies for sedentarism and for locomotion and surveillance, respectively). Fecal samples were analyzed for biweekly periods by 16S rRNA gene amplicon sequencing to profile bacterial composition and predicted functional pathways, and significant associations were found for each of the behavioural phenotypes. Both breeding station and local environment influenced gut microbial communities and personality profiles, underscoring the influence of management practices and local habitat in shaping the microbiome-behaviour nexus. Specific bacterial taxa and metabolic pathways were consistently associated with each behavioural phenotype, suggesting that microbial fecal signatures could serve as non invasive biomarkers for individual personality monitoring. This work constitutes the first comprehensive, multi layered examination of the interplay among behaviour, gut microbiota, and environmental factors in a large, wild carnivore. This integrative approach may help conservation programmes to optimize management decisions and improve reintroduction success

The gut microbiota is crucial for immune development and overall health in chickens. In commercial production, birds routinely receive multiple vaccines during early life. While individual vaccines are known to affect microbial composition, the impact of complex, multi-vaccine programs, as used in the poultry industry, is not well understood. This longitudinal study examined the impact of multiple live and inactivated vaccines, given at commercially relevant times from an early age, on gut microbial diversity and composition in layer chickens. We characterised microbiota profiles using 16S rRNA gene sequencing at pre- and post-vaccination timepoints across different vaccine groups. Overall, microbial diversity remained stable across most vaccines, indicating strong resilience of the gut microbiota to repeated immunological interventions. Differential abundance analyses identified changes in selected bacterial taxa following vaccination, with responses varying among vaccine groups. Notably, these changes were not sustained, as the gut microbial community returned to a stable state after the vaccination schedule. These findings underscore the robustness of the chicken gut ecosystem and lay a foundation for future research into microbiome-vaccine interactions and their implications for poultry health, immunity, and production efficiency.

Environmental change exposes ecosystems, including host-associated microbiomes, to stressors that occur repeatedly and in sequence, yet it remains unclear whether prior stressor history conditions host-microbiome responses to later perturbation. We used adult zebrafish (Danio rerio) to test whether sequential exposure to antibiotics, heat stress, the intestinal nematode Pseudocapillaria tomentosa, or pairwise stressor combinations altered gut microbiome structure, intestinal host gene expression, and host health outcomes. Across eight exposure regimes, prior stressor history and parasite exposure were associated with gut microbiome composition, while increasing prior stressor history was associated with reduced gut microbial diversity and convergence in community composition. Host intestinal transcriptional responses to parasite exposure were historically contingent, with parasite-associated differential gene expression varying non-linearly across prior stressor histories. Cumulative mortality increased with prior stressor history, whereas infection prevalence among surviving hosts decreased. Integrating microbial abundance, host gene expression, mortality, and neutral-community modeling identified Cetobacterium, Culicoidibacter, Flavobacterium, and Shewanella as candidate host-linked taxa associated with host response and survival. Collectively, these findings indicate that prior environmental stressor history shapes vertebrate host-microbiome responses to future perturbation and highlight specific gut microbial members as potential biomarkers or functional targets for follow-up studies.

The respiratory microbiome plays important roles in poultry health, immune regulation, and pathogen resistance, yet its development throughout the broiler production cycle remains poorly understood. This study investigated temporal changes in the tracheal microbiome of broiler chickens across production phases. Tracheal samples were collected during the starter (day 12), grower (day 21), early finisher (day 26), and late finisher (day 35) phases and analyzed using 16S ribosomal RNA gene sequencing. Tracheal microbial richness, diversity, community structure, and taxonomic composition changed significantly across broiler production stages, including starter, grower, early finisher, and late finisher feeding phases. Alpha diversity increased progressively throughout production, with significant increases in richness, diversity, and phylogenetic diversity during later stages. Beta diversity analysis revealed distinct microbial communities associated with each production phase, with starter-phase samples clearly separated from later phases. Taxonomic profiling showed dominance of Proteobacteria during the starter and grower phases, with enrichment of Methylobacterium-Methylorubrum and Pseudomonas during the starter phase and of Escherichia-Shigella during the grower phase. In contrast, the finisher phases exhibited reduced Proteobacteria abundance and increased Firmicutes and Actinobacteriota, including Lactobacillus, Ligilactobacillus, Faecalibacterium, Streptococcus, Staphylococcus, Romboutsia, and Corynebacterium. Overall, the tracheal microbiome underwent progressive maturation, shifting from a Proteobacteria-dominated community to a more diverse, complex, Firmicutes-rich ecosystem. These findings provide new insights into the development of the respiratory microbiome in broiler chickens and may support strategies to improve poultry respiratory health. Because dietary transitions occurred concurrently with age progression, the observed microbiome shifts should be interpreted as production-stage-associated changes rather than diet-specific effects.

Industrialization has reshaped human gut microbiomes, but its effects on other human-associated mammals remain poorly understood. Domestic dogs provide an informative comparative system because they have shared human environments and food systems for millennia yet retain distinct host biology. However, most canine microbiome studies have focused on industrialized companion animals, limiting our understanding of the ecological range of the domestic dog gut microbiome. We analyzed fecal 16S rRNA gene profiles from 261 dogs sampled across Nepal, Thailand, the United Arab Emirates, and the United States, spanning forager, agrarian, pastoralist, urban, and industrialized lifestyles; 257 dogs remained after excluding recent antibiotic exposure. Lifestyle was the strongest measured correlate of canine gut microbiome composition, and this structure persisted in restricted analyses of mature, non-shelter dogs sampled from temperate climate regions. Industrialized dogs differed from non-industrialized dogs through directional genus-level turnover, restructuring of VANISH- and BloSSUM-like microbial guilds, and shifts in predicted functional potential. Non-industrialized dogs were not microbiologically uniform: pastoralist dogs carried non-industrialized microbiome profiles but diverged from a simple forager-to-industrialized continuum. Cross-species comparisons with humans sampled across matched lifestyle categories showed parallel lifestyle-associated restructuring in both hosts, but host species remained the dominant axis of variation and the genera responding to industrialization were largely host-specific. These findings expand the ecological baseline for the domestic dog gut microbiome and identify industrialization as a major axis of microbiome restructuring in a long-term human-associated mammal. More broadly, they show that shared lifestyle transitions can impose parallel ecological pressures across host species without overriding host-specific community assembly.

Although the roles of host-associated microbiomes in animal health are increasingly recognised, the factors influencing their variation remain understudied. The relatively simple microbiome of honey bees is a relevant system to address this gap. In particular, the relationship between variations in microbiome composition and the ectoparasite Varroa destructor, the main threat to honey bee health worldwide, is poorly established. In this study, we used metagenomic and statistical analyses of 1442 European honey bee colonies to investigate the relationships between the honey bee microbiome, temporality, location, V. destructor load, and behavioural response to its infestation by the host. While season, year, and location were identified as the main drivers of microbiome variation, V. destructor load emerged as a significant factor associated with microbiome variation. Notably, we identify several pathogens and opportunists that correlated positively with V. destructor load, while the core symbiont Bombilactobacillus correlated negatively. This is compatible with a shift in the microbiome toward dysbiosis, which may be driven by or promote V. destructor parasitism. By contrast, we found only limited evidence of an association between the microbiome and resistance behaviours of the host against this parasite. While the study cannot establish causal relationships, we present the largest metagenomic analysis of honey bee microbiomes to date, providing robust, generalisable evidence about the factors driving variation in the microbiome composition of this ecologically and economically important pollinator. These findings may serve as additional markers in selective breeding programs targeting V. destructor resistance, which could ultimately improve honey bee health.

On their own, probiotics do not entirely eliminate pathogens during infection and disease. Instead, they often serve as adjuncts that rely on distinct mechanisms to reduce the presence of harmful bacteria. Our prior research indicates that an isolated Aeromonas dhakensis strain A603 kills pathogenic Vibrio strains through two antibacterial mechanisms: the type VI secretion system (T6SS) and phenazine (AdPhen). Here, we investigate A603 as a standalone probiotic for a shrimp disease model. This study shows that A603 prevents mortality from acute hepatopancreatic necrosis disease (AHPND) in shrimp caused by pathogenic Vibrio spp. that produce the PirAB toxin. AHPND infection alters the shrimp microbiota by increasing pathogen abundance and decreasing beneficial bacterial abundance prior to death. As both a prophylactic and treatment, A603 removes pathogenic Vibrio from shrimp and reverses such alterations in the microbiota using both T6SS and AdPhen. Collectively, our findings show that A603 antibacterial mechanisms prevent AHPND.

Anthelmintic resistance in cyathostomins is escalating worldwide, threatening equine health and highlighting the need for sustainable, ecology based parasite control strategies. Chicory (Cichorium intybus, Puna II) has emerged as a promising antiparasitic forage, yet its broader effects on the equine holobiont, parasites, microbiota, and host physiology remain poorly understood. We conducted a 32 day longitudinal grazing trial in young horses to assess how chicory affects parasitological outcomes, gut microbial ecology, nemabiome composition, behaviour, and host physiological and immune responses. Twenty-six naturally infected Anglo-Arabian horses were monitored weekly, with 13 grazing a chicory-based sward and 13 grazing a permanent pasture. Clinical parameters, body weight, and serum biochemistry remained stable across treatments, indicating that chicory was well tolerated. Immune profiles showed limited variation, although IL 10 increased in chicory fed horses, suggesting subtle immune modulation. Behavioural observations revealed no signs of discomfort and indicated slightly enhanced social interactions in the chicory group. Chicory grazing produced a marked reduction in cyathostomin egg excretion, accompanied by species specific shifts in nemabiome composition. Several cyathostomin taxa, including Cylicocyclus ashworthi, C. leptostomus, and C. nassatus, declined in chicory fed horses, whereas certain Cylicostephanus spp increased, indicating differential sensitivity rather than uniform suppression. Concomitantly, chicory induced profound ecological changes in the gut microbiota, including reduced alpha diversity, increased beta dispersion, and destabilised individual microbial trajectories. Several bacterial lineages, particularly Oscillospiraceae, Clostridiaceae, Lachnospiraceae, and Bacteroidales, were differentially enriched, reflecting a functional reorganisation of the intestinal ecosystem. Together, these findings demonstrate that chicory reduces parasite fitness, reshapes nemabiome composition, and alters gut microbial ecology while maintaining host physiological stability. Chicory thus emerges as a promising ecological tool for parasite control, capable of modulating the equine holobiont in ways that complement and potentially reduce reliance on conventional anthelmintic strategies. However, because its effects on gut microbial ecology remain uncertain, and may include shifts resembling dysbiosis, future studies are needed to monitor microbial dynamics more closely and clarify the long term ecological consequences of chicory grazing.

Animal microbiomes are shaped by both environmental exposure and host-associated filtering, but the relative importance of these processes remains poorly understood. Dung-associated insects provide an ideal model because they develop and feed in highly dynamic microbial environments. We investigated the gut microbiomes of six sympatric dung fly species of the genus Sepsis (Diptera: Sepsidae) and compared them with microbial communities in cow dung throughout a growing season in Switzerland. Using full-length 16S rRNA gene sequencing (PacBio), we characterized bacterial communities from 74 fly and 15 dung samples. Seasonal variation was the strongest predictor of microbiome composition, whereas host species exerted weaker effects that persisted after removing dung-associated taxa, indicating that gut communities are not merely passive reflections of environmental exposure. Only few gut microbiome reads were attributable to dung-associated taxa, and environmental overlap differed among fly species rather than season. A highly non-random core microbiome persisted across all six species: 36 bacterial genera (of 469) were shared by all hosts at [~]119-fold enrichment above random expectation and remained after removing dung-associated taxa. These findings support a two-layer model of microbiome assembly, in which seasonal environmental variation determines microbial availability while host-specific processes selectively retain a subset of taxa.

Bees and wasps are ecologically vital, but many species are declining due to anthropogenic stressors. Social bees harbour host-specific and dense gut microbiomes that affect their resilience to stress. However, there are tens of thousands of other bee and wasp species that vary in sociality and diet (including pollen-feeding and predatory guilds), traits known to influence host-microbe symbioses. The role of gut microbes in the biology of these species is largely unknown. Here, we measured the composition and absolute abundance of bacterial communities in adult abdomens across 61 genera and 14 families of field-collected bees, predatory wasps, and pollen wasps. We found that solitary bees and both wasp guilds harbor distinct bacterial taxa and lower bacterial abundances as compared with social bees. Bacterial abundances also varied extensively among and within genera of solitary bees, with little variation explained by body size, diet breadth, or nesting ecology. Further, microbiome composition was only weakly differentiated among solitary bees and the two wasp groups, even comparing herbivorous (pollen-feeding) and carnivorous taxa. We suggest that the sparse and somewhat stochastic microbiomes of solitary bees and wasps reflect weak host dependence on microbially mediated functions, a trait that may influence their responses to environmental change.

Background Colonisation resistance provided by the gut microbiota is a critical barrier to pathogen invasion, yet its study in vivo is constrained by the complexity and cost of vertebrate models. Here, we developed a humanised Galleria mellonella infection model by inoculating wax moth larvae with complex human faecal microbiota. 16S rRNA gene sequencing confirmed stable, reproducible establishment of a diverse human associated community across larvae over four days. Results Humanised larvae exhibited colonisation resistance against Salmonella enterica serovar Typhimurium, with mortality reduced to 20% compared to 90% in non colonised controls. To test whether prophages could overcome this barrier, we infected larvae with isogenic S. Tm strains differing in the presence of prophage P22. Infection with the P22 carrying strain resulted in a threefold higher larval mortality (60% vs. 20%), increased pathogen load, and a significant reduction in the abundance of resident E. coli. Free P22 virions were detected early after infection, indicating extensive prophage activity. Notably, P22 can neither adsorb nor lyse resident E. coli, indicating that prophage mediated invasion success did not rely on direct lysis. Instead, using high throughput metabolic profiling paired with whole genome sequencing of three replicate lineages, we found that phage activation intensified resource partitioning, accelerating functional metabolic adaptations in E. coli that significantly reduced the niche overlap between the invading pathogen and the commensal E. coli. Conclusion Our findings establish the first humanised G. mellonella model supporting complex human microbiota and provide a novel non lytic mechanism by which prophages influence species interactions. This scalable, low cost model offers a new platform to dissect pathogen phage microbiota interactions relevant to human gut ecology.

BACKGROUNDMetabolic dysfunction-associated steatotic liver disease (MASLD) is a systemic immunometabolic disorder rapidly increasing worldwide, affecting nearly 38% of adults. Gut dysbiosis and host genetic factors, such as PNPLA3 I148M variant, modulate disease development and progression. Through the gut-liver axis, increased intestinal permeability enables microbial translocation to the liver, promoting inflammation and metabolic disruption. However, the composition and functional potential of the hepatic microbiome remain poorly characterized. Understanding its relationship with histological injury and genetic susceptibility may provide novel mechanistic insights. We hypothesized that the hepatic microbiome composition and function are associated with histological severity and PNPLA3 genotype in this disease. AIMTo characterize the hepatic microbiome and assess its association with histological severity and PNPLA3 genotype. METHODSThis cross-sectional observational study included 30 patients with MASLD from a tertiary care hospital. Liver tissue underwent shotgun metagenomic sequencing. Histological severity was assessed using the NAFLD Activity Score (NAS). PNPLA3 genotype was determined by PCR. Differential abundance and functional enrichment analyses were performed using MaAsLin2. Somatic variants were identified using Mutect2. Correlation networks were constructed using Spearmans correlation coefficients. RESULTSPatients with advanced histological injury (NAS [≥]5) and PNPLA3 I148M carriers showed a trend toward higher somatic mutational load and a markedly reduced microbial abundance. Analyses revealed broad compositional shifts across bacterial, fungal, viral, and eukaryotic taxa, affecting both commensal and context-dependent pathobiont lineages. Pseudomonas species were enriched, whereas Siphoviridae phages were depleted in advanced disease and PNPLA3 I148M carriers. Functional analysis revealed enrichment of pathways related to nutrient transport and metabolic stress adaptation, while TonB-associated functions were enriched in advanced liver injury but depleted in PNPLA3 I148M carriers. Network analysis identified Sphingomonas leidyi as a keystone node associated with hexosamine metabolism. Salmonella enterica abundance positively correlated with somatic variant burden, suggesting a link between microbial signatures and genomic instability. Histological progression and the risk PNPLA3 genotype were accompanied by marked topological simplification, reflecting less resilient community structures. CONCLUSIONSThe hepatic microbiome in MASLD is a low-biomass, polymicrobial ecosystem shaped by the host genetic background. Its functional activity, taxonomic composition and system architecture bidirectionally relate to liver DNA instability and the severity of histological damage. Core tipThis study characterizes the multi-kingdom hepatic microbiome in MASLD using FFPE-derived metagenomics. We demonstrate that microbial abundance-including bacteria, fungi, protozoa, and viruses- significantly decreases with increased histological severity and the PNPLA3 risk genotype. Rather than global diversity shifts, results showed that disease progression could be linked to specific functional adaptations and simplified microbial network connectivity. In addition, we described associations between specific taxa and somatic mutational burden, suggesting a link between microbial signals and genomic instability. These findings indicate that changes in the liver microbiome as a whole, rather than specific taxonomic modifications, influence MASLD pathophysiology.

Predators are often considered regulators of disease in prey populations, a concept central to the "healthy herd hypothesis". This hypothesis suggests that by preferentially removing infected individuals, predators can reduce parasite prevalence. However, predators may also act as disease vectors, facilitating the spread of parasites. We investigated whether stickleback fish (Gasterosteus aculeatus) can act as vectors for the transmission of the obligate bacterial parasite Pasteuria ramosa to its Daphnia host, a widespread freshwater zooplanktor. We fed infected D. magna to sticklebacks, and subsequently analysed faecal samples for the presence, viability, and infectivity of parasite transmission stages (= spores). We recovered approximately 60% of the consumed spores from fish faeces and these spores did not suffer from reduced infectivity to D. magna. Additionally, spores associated with sloppy feeding did not reduce infection rates. Thus, consumption of infected hosts by fish does not eliminate the parasite, but in contrary, may contribute to the spread and persistence of P. ramosa in natural populations, potentially influencing parasite dynamics in natural freshwater ecosystems.

Predatory bacteria such as Bdellovibrio are emerging as ecological modulators in microbial communities by restructuring community composition, yet their roles in host-associated microbiomes remain poorly understood. Using Caenorhabditis elegans as model host and its defined microbiota, we investigated how two Bdellovibrio strains with distinct prey ranges (B. tiberii MYbb2 and B. krueschi MYbb4) affect microbial community composition and host life-history traits. Both strains consistently altered microbiome composition, with MYbb4 causing more pronounced alpha-diversity shifts and MYbb2 selectively enriching strains of the genus Ochrobactrum which coincided with higher host median lifespan. Genome-based predictions indicate that de novo vitamin B12 synthesis by Ochrobactrum underlies the observed host phenotype, which was confirmed through quantitative measurements of the vitamin in mono-cell cultures. Employing the acdh-1p::GFP transcriptional reporter strain, we confirmed that a diet of B12-producing bacteria suppresses the B12-independent propionate detoxification pathway in the host, demonstrating that bacterially produced B12 is bioavailable to C. elegans. Exogenous B12 supplementation assays further confirmed the lifespan-extending effect. Together, these results suggest that predation-driven enrichment of B12-producing bacteria maintains B12 levels sufficient to detoxify propionyl-CoA via the B12-dependent pathway, preventing the accumulation of toxic metabolic byproducts that would otherwise arise under B12-limiting conditions and reduce host lifespan. Our findings demonstrate that predatory bacteria are important drivers of microbiome structure with direct consequences for host physiology, representing an underappreciated ecological mechanism for microbiome modulation.

The guano of insectivorous bats holds ecological information that can be assessed non-invasively to characterize the gut microbiome and diet, alongside environmental microbes and pathogens of conservation concern. Despite this potential, how guano communities change during decomposition remains understudied, particularly inside anthropic roosts rather than caves. In this study, guano from little brown bat (Myotis lucifugus) colonies was sampled monthly across the summer maternity season from three sites across two locations in Connecticut, USA, at fresh deposition and at 4, 8, and 12 weeks following deposition. Resolving these cross-kingdom signals required five workflows: short-read 16S and ITS2 amplicon sequencing (Illumina) for bacterial and fungal profiling, long-read CO1 metabarcoding (Oxford Nanopore) for arthropod diet in fresh samples, long-read shotgun metagenomics for viral identification in aged samples, and targeted qPCR for organisms of bat, human, and forest-health concern. Fresh guano generated a consistent bacterial signal across sites, whereas fresh fungal communities differed by site. Responses to decomposition depended on roost setting: exterior sites lost fungal diversity and shifted toward environmental aerobes over time, while the interior roost retained the fresh sample profile. Dietary composition varied temporally, was dominated by Diptera, and included the invasive emerald ash borer (Agrilus planipennis). Pseudogymnoascus destructans, the causal agent of white-nose syndrome, occurred in fresh and aged samples at all three sites but persisted for 12 weeks only at the interior roost, where antifungal bacterial taxa were depleted. Long-read shotgun metagenomics of aged guano recovered roughly 100 viral species, predominantly bacteriophages, alongside non-bacteriophage mastadenoviruses associated with humans, bats, and other mammals. These results show that anthropic structures influence the trajectory of guano microbiome succession, and that maternity colony guano enables non-invasive assessment of environmental pathogens, bat diet, and bacterial and fungal communities.

Evolutionary and demographic processes such as selection, drift and migration shape the genetic variation of populations. Genetic diversity is often lower in populations toward higher latitudes. This decrease potentially threatens their survival as several factors are putting more pressure on the populations, including the spread of infectious diseases. In this study, we combined whole-genome re-sequencing with MHC class II genotyping and skin microbiome profiling in Bufo bufo and B. spinosus, two closely related European toad species. We investigated the underlying immunogenetic and microbial variation resulting from different demographic histories and environmental conditions to identify their potential impact on infection outcomes in these two species. We found lower immunogenetic diversity in B. bufo compared to B. spinosus, with highly significant differences in genes related to adaptive and innate immunity. We found lower overall MHC class II diversity and skin microbiome diversity at the species level in B. bufo, compared with B. spinosus. In contrast, at the individual level, B. bufo showed higher MHC allelic diversity and greater diversity in the core skin microbiota than B. spinosus. Together, our findings suggest that divergence in immunogenetic background and host-associated microbial communities may underlie differences in susceptibility to emerging infectious diseases. This integrative framework provides new insight into how host genetics and microbial communities jointly influence disease outcomes across environmental gradients.

The aim of this study was to evaluate the impact of two sustainable dietary protein sources on the structure and composition of the gut microbiota in European sea bass (Dicentrarchus labrax) juveniles. These protein sources were incorporated to the aquafeeds containing (a) Lupinus albus meal, treated with either exogenous enzymes (Solid state hydrolysis-SSH) or fermented with Saccharomyces cerevisiae (Solid state fermentation, SSF) and (b) Lagocephalus sceleratus meal. In the first case (a), the control aquafeed simulated a standard commercial diet, containing soybean meal whereas in the rest of the diets soybean meal was partially or totally replaced by hydrolysed or fermented Lupin meal. In the second case (b) the fish were fed Lagocephalus sceleratus unprocessed fishmeal as well as treated at different temperatures to deactivate tetrodotoxin (TTX). A control diet with 30% commercial fish meal was also fed as a reference diet. Both diets in all inclusion levels did not cause any significant gut microbiota change, suggesting their neutral role in this aspect. However, the gut bacterial communities of the fish fed with 12.5% lupin meal inclusion, had increased amino acid biosynthetic pathways suggesting a beneficial effect.

Pre-harvest litter management is a key determinant of broiler production conditions, influencing NH3; generation, pathogen exposure, nutrient retention, and microbial reservoirs that accumulate across production cycles. Conventional chemical and physical management strategies can support flock health, but their effects on pathogen-associated bacterial populations are often transient and may not account for the microbial interactions that govern persistence, exclusion, and community succession. Here, we evaluated an alternative litter management strategy combining IndigoLT pre-/postbiotic with reduced-rate NaHSO4; across two broiler growouts, with litter sampled at the end of each flock to determine impact on prokaryotic microbiome structure, physicochemistry, and Enterococcus abundance. Alternative management influenced observed richness, phylogenetic diversity, community composition, and co-occurrence network structure while reducing the relative abundance of Enterococcus, including E. cecorum and E. hirae. Digital PCR corroborated sequencing-based Enterococcus abundance patterns, although 16S-based treatment effects were not always reflected as lower absolute copy number at terminal sampling, consistent with reduced proportional dominance rather than sustained absolute suppression. Complementary biofilm- and growth-inhibition assays performed with IndigoLT demonstrated context-dependent antibiofilm and bacteriostatic activity against reference and poultry-derived Enterococcus isolates, with stronger responses for E. cecorum than E. hirae and bactericidal-level reductions in viable recovery at higher exposure levels. These findings demonstrate that biologic-based litter management can alter microbiome structure and pathogen-associated taxa under commercial production conditions, providing a basis for microbiome-informed amendment strategies aimed at reducing pathogen load and supporting broiler health.

Biofilms on aquaculture infrastructure harbor diverse microbial communities that may influence water quality and fish health, yet the temporal dynamics of these communities remain poorly characterized. Here, we used 16S rRNA gene amplicon sequencing to profile biofilm communities on concrete raceway surfaces across an 80-day rainbow trout (Oncorhynchus mykiss) indoor hatch-house production period. One hundred twenty-three wall swab samples from 19 raceways at six time points (9, 23, 38, 53, 65, and 80 days) were analyzed after stringent quality control. Beta diversity analyses revealed that biofilm communities at each time point were significantly distinct (PERMANOVA, p < 0.001 for all pairwise comparisons), with early communities exhibiting greater variability than late-stage biofilms. Total bacterial load increased approximately 2.5-fold from early to late stages (qPCR, p < 0.001). Differential abundance testing (ANCOM-BC) identified 57 differentially abundant genera between early-and late-stage biofilms, and random forest classification distinguished early from late communities with over 93% test accuracy. A clear successional trajectory emerged: early biofilms were dominated by pioneer taxa including Pseudomonas, Caulobacter, and Flavobacterium; mid-succession communities featured predatory Bdellovibrio and the methylotroph Methylotenera; and mature biofilms were enriched in saprophytic Saprospiraceae and Haliscomenobacter, polysaccharide-degrading Verrucomicrobiaceae, and cooperative predatory myxobacteria. Flavobacterium columnare, a pathogen of concern in aquaculture, was detected at low levels throughout the production period. These results demonstrate predictable ecological succession in freshwater built environment biofilms and provide a foundation for understanding the role of surface-associated microbial communities in hatchery management.