Microbial Ecology

Microbial community assembly on leaves (the phyllosphere) is influenced by deterministic, stochastic, and historical processes. However, the importance of historical processes such as priority effects, where the timing and order of colonization influence community assembly, has not been extensively investigated in field-based phyllosphere studies. We designed a field experiment in a temperate forest in Quebec where we inoculated five strains of Methylobacterium onto the leaves of red maple and American beech trees, either early or late during the growing season. We quantified microbial community structure throughout the season using metabarcoding of a Methylobacterium-targeting rpoB primer as well as a 16S rRNA primer targeting the entire bacterial community. We show that the effects of inoculation on bacterial communities depend on inoculation timing: early-season inoculation led to strain-specific shifts in community composition, while late-season inoculation had no significant effect. Although the effect of early-season inoculation weakened over time, early- and late-season inoculation still led to different community compositions at the end of the growing season. Our study demonstrates that priority effects contribute to the assembly of phyllosphere bacterial communities under field conditions, with arrival timing and strain identity shaping community trajectories. Considering historical processes will thus be crucial for successful microbiome management.

Megasphaera elsdenii is best known as a prominent lactate consumer within the rumen microbial community in livestock, and its metabolic properties are relatively well studied. In humans, it can be isolated from healthy donors feces and, more often, from patients feces with diverse inflammatory conditions. It is currently unclear whether human and animal gut isolates are genetically related and perform the same metabolic function. In this study, we compared 86 M. elsdenii genomes from human feces (as a proxy for the human gut) to animal gut isolates and contrasted both with phylotypes colonizing the vaginal niche. Phylogenetic analysis showed that human gut samples form a genetically homogeneous branch with animal gut isolates, with samples intermingled and no evident host-specific clustering. Human gut samples shared most of their genes and biochemical pathways with isolates from swine and cattle, despite differences in their digestive tracts. When human and animal samples were labeled, supervised analysis detected only minor differences in pathway content. Genome-scale metabolic modeling suggests that human and animal gut samples likely share identical carbon and energy source requirements. Moreover, the requirements for lactate and acetate were conserved across all studied samples, regardless of the host or niche. Finally, we found no virulence genes, and lactate utilization remains a plausible explanation for M. elsdenii accumulation in different niches. IMPORTANCEMegasphaera elsdenii is considered a commensal in the human gut and animal rumen. However, M. elsdenii tends to be more abundant in patients feces with diverse inflammatory conditions. As we know little about strain diversity and biology in human isolates, comparison with better-studied isolates can be informative. In this study, we compared M. elsdenii genomes from the human gut to those in better-studied isolates from ruminal and non-ruminal animals. Human-derived samples from patients and healthy donors were genetically very similar to animal isolates and may have shared a common origin. We found that human and animal samples have a similar genomic makeup and metabolic potential, and that neither group harbors virulence genes. We hypothesize that M. elsdenii is a benign commensal that grows in response to lactate accumulation in the inflamed gut.

Microorganisms, found in every environment, play an important role in recycling matter and providing energy to the ecosystem. Here, we used 18S and 16S metabarcoding to study bacteria, cyanobacteria and protist community diversity and functional ecology among different environments of typical lake, river, marine and soil from the Yangtze Delta of China. The results showed that the similarity of cyanobacteria and protozoa communities in soil and aquatic environments was both higher than that of bacteria and microalgae communities. While the diversity of cyanobacteria and bacteria in lake was higher than that in river, marine and soil, the diversity of microalgae and protozoa in river was higher than that in lake, marine and soil. The {beta} diversity of cyanobacteria and bacteria marked differences in lake from other environments. But The distribution of dominant families of cyanobacteria, bacteria, microalgae and protozoa is similar to diversity. While significant positive correlations were found among dominant species of cyanobacteria, protozoa, bacteria and microalgae in lake the dominant species among bacteria, microalgae and protozoa in river, marine and soil all showed more negative correlations. Our study provides the basis for understanding the functional ecology of microbes in the micro-food webs of different environments.

Like for many other organisms, earthworms host a microbiota consisting of bacteria, fungi and other micro-organisms originating from their surrounding environment. In earthworms, these microbes, either associated with the gut, skin, kidney-like nephridia, gizzard or cocoons, aid their hosts with the digestion of organic matter, immune responses against pathogens, and/or detoxification. Unfortunately, pollution is an increasing threat to soil health and soil biodiversity, and numerous articles have characterized both the microbial communities associated with earthworms, as well as the effects of soil pollution on these microbial communities. Here, we aim to synthesize data from across 159 articles to identify consistent global patterns and gaps in the study of earthworms' microbial communities. Additionally, we combined 16S rRNA metabarcoding data from almost 30 articles in a meta-analysis to evaluate the changes in the diversity of earthworm's and surrounding soil's bacterial microbiota after pollutant exposure. Through an evidence map, we clearly showed that most of the earthworm microbiota data came from the Eisenia fetida species reared in laboratory conditions, and only a handful of studies sequenced fungi (ITS) and protists (18S rRNA) in addition to bacteria and archaea (16S rRNA). The scarcity of studies from wild populations, environmental conditions, and the low microbial diversity targets and sources, cannot support a comprehensive view on earthworm's microbial ecology. Nonetheless, results of the meta-analysis indicate that the data did not support the occurrence of significant changes in the microbiota richness and diversity as result of pollution exposure. However, the microbial communities altered their composition, becoming more dissimilar. We suspect the microbiota response to pollution to be host-species specific, but the lack of data severely limited our ability to further elucidate the effect of pollution on earthworm microbiota.

Bacteria inhabiting leaf surfaces - the phyllosphere - are crucial to plant health and ecosystem functioning. Methylobacterium is a taxonomically diverse, growth-promoting genus, ubiquitous on leaves. Different plant species host distinct Methylobacterium communities, but the genomic and functional basis of Methylobacterium symbiotic associations with particular host species remains poorly understood. Here, we used a metapangenomic approach to quantify the influence of host species on Methylobacterium assemblages, to identify genes potentially involved in Methylobacterium adaptations to host species, and to evaluate the contribution of Methylobacteriums accessory pangenome to these adaptations. We sequenced the metagenomes of 25 phyllosphere communities spanning five host species in a temperate forest in Quebec, Canada, and mapped these metagenomes onto Methylobacteriums pangenome to obtain nucleotide-level coverage and composition for each population on each individual host. We revealed strong divergences in the species- and gene-level community structure of Methylobacterium, driven by host phylogeny and plant form. Conifer communities were notably enriched in genes involved in amino acid, lipid, and carbohydrate metabolism; broadleaves, in genes involved in cell membrane, signalling, defense, and chemotaxis; and trees, in genes related to photosynthesis, oxidative phosphorylation, and translation. The shrub Corylus cornuta was a reservoir of Methylobacterium taxonomic diversity, and harboured numerous accessory genes under positive selection. Methylobacteriums accessory pangenome, evolving under weaker purifying selection, contributed importantly to gene-host associations, supporting its adaptive role. By linking genes to phyllosphere niches, our study shed light on the genetic basis of host adaptation and highlighted the crucial role of forest biodiversity in shaping microbial ecology and evolution.

Entomopathogenic Nematodes (EPNs) have a great potential to control various insect crop pests, but their ability to perform their entire life-cycle in insects depends on environmental conditions. The insect killing ability of the endosymbiotic bacterial species (i.e., Xenorhabdus) located in the EPNs' gut (Steinernema) has been described for years. More recently, experimental data have suggested that other bacteria species of the nematode microbiota may also contribute to the parasitic success of the EPNs. EPNs and their associated bacteria should therefore be considered as holobionts. Global change, among its many predicted consequences, is expected to increase temperatures which may impair the parasitic success of these EPNs in the insect. Furthermore, higher environmental temperatures could modify the composition of the EPN-associated microbiota and could therefore also contribute to parasitic impairement. Using a S. carpocapsae strain originating from a French orchard, we investigated these hypotheses by experimentally evolving independent EPNs lineages in vivo in the lepidopteran species Galleria mellonella, at elevated and control temperatures. Several life-history traits of the evolved lineages were measured (insect killing ability, EPN emergence rate, EPN reproduction rate), and the bacterial microbiota of the evolved lineages was characterized by a metabarcoding approach. The EPNs' lineages evolved at higher temperature showed impaired emergence rate and their reproduction rate was lower, when compared to lineages evolved at control temperature. The bacterial microbiota composition of these lineages was modified, with particular enrichment in bacteria of the genera Alcaligenes and Pseudomonas. Our results provide new evidence that modification of the environment (here, the abiotic parameter temperature) during several successive generations can modify the parasitic success of EPNs, concomitantly to a change in the microbiota composition. A better understanding of the life-history traits of these EPNs should help improve the biocontrol efficiency in a context of global warming.

Sponges are among the earliest-diverging metazoans, and their evolutionary success has been strongly linked to their symbiosis with microorganisms. While marine sponge-microbiome associations have been extensively characterized, freshwater sponges remain comparatively understudied, particularly in tropical systems such as the Amazon, where sponges undergo seasonal flood pulses and urbanization-derived disturbance. Here we characterized the microbiomes of two freshwater sponge species, Drulia brownii and Tubella paulula, from two contrasting sites in the Tapajos River (Amazon basin), a non-urbanized and an urbanized site, at two time points, the rainy and dry seasons. The whole metagenome was sequenced using a long-read shotgun approach, and metabarcoding was employed to characterize the gemmule microbiome. The analyses revealed that microbiome composition was primarily influenced by season, with urbanization exerting a secondary but significant effect. In non-urbanized rainy-season sponges, the microbiome was dominated by Pseudomonadales and Bacillales, whereas urbanized sponges showed more diverse profiles enriched with Burkholderiales, a possible symbiont. During the dry season, communities converged across sites, with Burkholderiales becoming the dominant taxon, including in gemmules. The 23 high-quality MAGs recovered revealed symbiosis-related genes, broad biosynthetic repertoires, and a distinctive carbohydrate-active enzyme profile. Functional analyses suggest that Pseudomonadales and Burkholderiales may play complementary roles across seasons, and Bacillales may be associated with organic matter turnover. These results show that Amazonian freshwater sponges harbor stress-sensitive but resilient microbiomes, with seasonality driving major compositional shifts and urbanization accelerating convergence toward Burkholderiales-dominated consortia, and highlight the central role of bacterial symbionts in nutrient acquisition, photoprotection, and chemical defense.

Prevotella melaninogenica is a Gram-negative bacterium that has been implicated in numerous disorders such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis, and despite its abundance in the human microbiome, there is still limited knowledge on the fundamental physiology describing Prevotella melaninogenica. To properly advance Prevotella biology, it is critical to report and document such characterizations of Prevotella; therefore, the purpose of this study is to examine and define the phenotypic and growth characteristics of P. melaninogenica ATCC 25845. In this study, we found that Prevotella melaninogenica ATCC 25845 is a Gram-negative bacterium that is predominantly short and medium rod-shaped, with occasional filamentous rods. SEM imaging revealed that this filamentation may serve as division centers, points from which multiple daughter cells form. We also describe the antibiotic profile, which showed susceptibility to four classes and resistance to six classes of antibiotics. Carbon source growth kinetic characterization revealed a preference for carbohydrate sources.

Recent years have witnessed the discovery of an immense diversity of protists in almost all ecosystems on Earth. However, a major challenge consists in identifying diversity patterns and the drivers that explain the distribution of this diversity. While several works have focused on local parameters, only a few studies have addressed this question at regional scales. In this study, we characterize the diversity and distribution of Arcellinida (testate amoebae) at regional scale of a climatically contrasted country, Bulgaria. We collected 75 samples geographically evenly distributed and representative of the country's natural ecosystems and used metabarcoding to determine their diversity. Communities composition differed markedly among habitats, revealing sharp beta diversity patterns. "Mesophilic" environments such as forests and ponds were most diverse, while acidic and oligotrophic Sphagnum peatlands were poorest and hosted the highest proportion of unique taxa. Our analyses of richness-environment based on a Random Forest approach applied to all soil systems indicated that stable humidity (low silt content, low precipitation seasonality) and intermediate soil carbon stocks had a positive effect on richness. The predicted increase in continentality in the next decades in Bulgaria calls for testing climatic models to evaluate the potential risk of declining Arcellinida biodiversity.

Many insect populations are experiencing thermal stress as a result of global change, making it imperative to investigate how their relationship with other organisms will be impacted by heat disturbances. Microbial symbionts, such as bacteria, have the potential to enhance or inhibit an insect's thermal tolerance. Solitary bee larvae host bacteria within their food stores ("pollen provisions"), which have been shown to benefit survival and development; however, it is unclear how heatwaves brought about by climate change will impact their relationships with these bacterial partners. In this study, we subjected blue orchard bee (Osmia lignaria) eggs and larvae to a 4-day heatwave (35 {degrees}C daytime:22 {degrees}C nighttime) or kept them at control temperatures (25 {degrees}C daytime:15 {degrees}C nighttime), then returned all bees to control temperatures for a 5-day recovery period. We assessed bacterial communities within pollen provisions and larval development stage pre-heatwave (Day 0), immediately post-heatwave (Day 4), and following the recovery period (Day 9). Bacterial community composition, diversity, and abundance were resilient to heat stress, but larval bees developed faster when subjected to a heatwave. This finding refutes the hypothesis that bacteria within pollen provisions modulate blue orchard bee responses to heat, suggesting instead that developmental effects could be more largely shaped by bee physiology or interactions with microorganisms other than bacteria.

Predatory protists are important in shaping terrestrial microbial ecosystems, but their roles in the phyllosphere, or the communities on aerial plant surfaces, are poorly understood. Previous work found that the order Colpodida dominated heterotrophic protist communities in the phyllosphere. While most protists were sporadically present, a few Colpodida variants were prevalent and abundant, indicating that these variants may represent species adapted to the phyllosphere. To identify these organisms, we cultured colpodids from field-collected tomato leaves and performed phylogenetic analysis of the 18S rRNA gene. Five of nine independent isolates matched the most prevalent Colpodida variant previously identified as leaf-enriched through amplicon sequencing, and these isolates comprised a novel clade of Paracolpoda steinii. When compared to a maize root isolate of Colpoda inflata, an abundant rhizosphere ciliate, a P. steinii isolate was similar in size and growth yield on E. coli, but grew to higher yields and formed large cyst clusters when incubated with model phyllosphere bacteria prey Erwinia and Pseudomonas. We developed and validated quantitative PCR (qPCR) methods for detection and cell abundance estimation of the P. steinii phyllosphere clade, C. inflata, and the order Colpodida in environmental samples. In inoculated greenhouse plants, qPCR-estimated protist populations matched measured inoculum levels, and protist inoculum was still detectable after five days. In an uninoculated tomato field, P. steinii was detected on all plants, with greatest abundances observed in lower leaves and after a rain event. P. steinii comprised up to 18.7% of total leaf Colpodida populations, which were estimated at up to [~]1400 organisms per gram of fresh weight. The findings demonstrate that Colpodida communities are consistently present on tomato leaves, dynamically affected by the abiotic environment, and include significant populations of P. steinii. We propose that the P. steinii isolates and qPCR tools presented can be used as a model system to investigate colonization and distribution patterns, biotic interactions, genetic adaptations, and agricultural applications of leaf predation.

Giant clams are some of the largest bivalve molluscs. They form a vital partnership with Symbiodiniaceae dinoflagellates that supply most of their energetic requirements. However, the factors that shape giant clam-associated photosymbiont communities remain unknown. Here, we profiled Symbiodiniaceae communities using ITS2 metabarcoding in eight giant clam species (Hippopus hippopus, H. porcellanus, Tridacna crocea, T. derasa, T. gigas, T. maxima, T. noae and T. squamosa) from 11 sites across the Philippine archipelago. Symbiodiniaceae community structure was shaped by an interplay between giant clam host and environment. Most giant clams were dominated by members of a single symbiont genus, with Cladocopium as the most prevalent, followed by Durusdinium and Symbiodinium. However, giant clam hosts also exhibited flexibility in their symbiotic partners that was evident across sites. Differences in giant clam-associated symbiont communities may contribute to differences in holobiont function and adaptability to variable environments. These findings deepen our understanding of giant clam-Symbiodiniaceae associations, offering a framework for predicting how giant clams may be affected by increasingly stressful reef conditions and, more importantly, informing strategies to improve mariculture and conservation practices.

Cattle harbor diverse bacterial communities across the gastrointestinal, respiratory, reproductive, mammary and other anatomical systems, but body-wide microbial biogeography remains poorly defined. To address this knowledge gap, we conducted a meta-analysis of publicly available bovine 16S rRNA gene amplicon sequencing data from 5,637 samples from 47 studies across six geographic regions and 27 anatomical sample types. Bacterial community structure differed significantly among sample types (PERMANOVA, R2 = 0.245, P = 0.0001), indicating spatial organization of bacterial communities across the bovine body, although study-level effects also contributed substantially to community variation. Communities were generally more similar within than between anatomical systems. Bacterial richness, diversity, taxonomic composition, and indicator taxa varied among sample types, with gastrointestinal, mammary-associated, ocular, and hoof microbiota exhibiting greater diversity than microbiota from liver, joint, and several reproductive samples. Distinct bacterial communities characterized the gastrointestinal, respiratory, reproductive, and mammary systems, as well as other anatomical sites. Despite these differences, several bacterial taxa were shared across multiple anatomical niches, particularly among male and female reproductive sites and among mammary-associated niches. This study provides a comprehensive body-wide characterization of bacterial biogeography in cattle and establishes a baseline for future studies of bovine microbial ecology and host-microbiome interactions.

Microbial communities play key roles in the transformation and cycling of elements ranging from required macronutrients to toxic metalloids. Next-generation sequencing has been applied across multiple ecosystems to probe the interplay of microbial community structure and functional potential with respect to elemental cycling. Shotgun metagenomics collects marker gene sequences without amplification and is costly for large numbers of samples and deep coverage. Conversely, amplicon sequencing of taxonomic marker genes, e.g. 16S and 18S rRNA, is cost-effective for large numbers of samples, but provides limited functional insight. A middle ground between the two approaches is needed to analyze community structure and functional potential within a sample while remaining cost-effective with high throughput. To address this need, we developed a standardized workflow for multiplexed amplicon sequencing from sample collection through data analysis for diverse sample types, including freshwater, sediments, and soils, that produces data and publication-ready figures for multiple taxonomic and functional genes for carbon, nitrogen, phosphorus, sulfur, and arsenic cycling for each sample analyzed. The workflows utility was shown by analyzing 11 taxonomic and functional gene amplicons sequenced from 25 samples with high technical replicate similarity. The workflow is named CAMASE for Compositional Analysis of Multiplex Amplicon Sequencing Experiments. This proof-of-concept shows that CAMASE economically produces standard amplicon sequencing outputs (ASV/OTU counts and taxonomy, PCA, and relative abundance plots) for hundreds of amplicon by sample combinations and provides specific recommendations for implementation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/741237v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@14c746corg.highwire.dtl.DTLVardef@6f9890org.highwire.dtl.DTLVardef@894038org.highwire.dtl.DTLVardef@d72b2a_HPS_FORMAT_FIGEXP M_FIG C_FIG Samples are collected in a preservative and material collected on filters prior to DNA extraction. Target gene amplicons are produced in parallel with internal barcodes enabling sequencing in a single run followed by compositional data analysis. All wet lab protocols, code markdowns, and templates for required metadata files are available at https://hansonlabgit.dbi.udel.edu/aprange/CAMASE. Created in BioRender. Bennett, A. (2026) https://BioRender.com/ymnojt0

Ticks are obligate hematophagous arthropods and feed exclusively on blood. As blood is nutrient-poor, ticks rely on bacterial endosymbionts to synthesise nutrients, yet the diversity and functional roles of these symbionts in Australian ticks remain largely uncharacterised. This is critical to address as these ticks are of high medical importance in Australia. In this study, shotgun metagenomic sequencing was performed on Bothriocroton concolor, Bothriocroton hydrosauri, Haemaphysalis longicornis and Ixodes holocyclus, enabling the recovery of six complete or partial metagenome-assembled genomes (MAGs). These comprised Coxiella-like endosymbionts (CLE), a facultative Rickettsia symbiont, and two Midichloria mitochondrii strains (Ixholo1 and Ixholo2). Functional annotation of these taxon-specific symbionts revealed the absence of virulence factors and the presence of B-vitamin and/or heme biosynthesis genes, indicative of nutritional mutualism, which is essential for tick hematophagy. The CLEs additionally harbour genes of the shikimate pathway, which modulate blood feeding in ticks by regulating serotonin biosynthesis. Furthermore, functional annotation and pangenomic analysis of Midichloria spp. found evidence that the genus may encompass multiple species, as well as the retention of genes potentially associated with an intramitochondrial lifestyle in M. mitochondrii Ixholo2. Tick microbiomes are dominated by non-pathogenic microorganisms, which are often overshadowed by pathogens. These include the endosymbionts, which can influence host biology and pathogen transmission, and are fundamental for the development of diagnostic tools and taxon-specific tick biocontrols.

The obscure life histories of many kickxellalean genera remain a bottleneck for comprehensive taxon sampling and phylogenetic reconstruction of the order. While Kickxellales has long been regarded as saprobes inhabiting soil or dung, the discovery of "amphibious fungi" such as Unguispora, which exhibits dimorphic growth between the animal gut and feces, suggests a cryptic gut-dwelling stage within these genera. Given its phylogenetic proximity to Unguispora, ecophysiological traits of Linderina were investigated to evaluate its potential association with the animal gut. Two isolates of L. macrospora were obtained from Japanese soil, representing the first record of this species in Japan. Physiological assays revealed that the optimal temperature for both vegetative growth and sporulation was 25-30 {degrees}C. Furthermore, comparative growth assays on different media demonstrated that sporocladium abundance per sporangiophore is sensitive to nutrient availability, and nutrient-poor media were determined to be the most suitable for evaluating morphological characterization. Under anaerobic, nutrient-rich conditions which are known to induce yeast-like growth in Unguispora, sporangiospores of L. macrospora produce arthrospores. Although marked morphological plasticity was observed during the arthrospore formation, the occurrence of yeast-like unicellular proliferation suggests a potential relationship with the animal gut. Additionally, vegetative growth and sporulation were markedly inhibited by white light exposure; notably, a lethal effect on growth was observed during incubation at 20 {degrees}C, indicating that the natural niche of the species is restricted to light-shielded environments. Our findings will help to elucidate the cryptic life cycles and evolutionary trajectories within Kickxellales.

Four rhizobial strains isolated from root nodules of tropical leguminous plants in Costa Rica were characterized. Strain LEGMi-135b was isolated from Lonchocarpus felipei root nodules, LEGMi-12c from Cojoba arborea, LEGMi-198b from Calliandra haematocephala and Rhizobium altiplani LEGMi-166a from Mimosa pigra. Phylogenetic analyses based on the 16S rRNA gene and four concatenated housekeeping genes (recA, atpD, GATase and gyrB) placed these strains within the genera Martinezella (LEGMi-12c, LEGMi-135b, and LEGMi-198b) and Rhizobium (LEGMi-166a). Genome-based analyses showed that strain LEGMi-135b exhibited an average nucleotide identity (ANI) value of 94% and a digital DNA-DNA hybridization (dDDH) value of 53.2% compared with its closest related species, Martinezella hainanensis CCBAU 57015T, supporting its classification as a distinct species. LEGMI-12c exhibited ANI and dDDH values of 98% and 87%, respectively, compared with Martinezella hainanensis CCBAU 57015T. LEGMi-198b showed the highest similarity to Rhizobium cerradonense CNPSo 3464, with ANI and dDDH values of 98% and 81.4%, respectively; however, this taxon has not been validly published. LEGMi-166a exhibited ANI and dDDH values of 100% and 99.3%, respectively, compared with Rhizobium altiplani BR 10423, confirming its assignment to this species. Additionally, differentiation of strains LEGMi-135b and LEGMi-198b from their closest phylogenetic relatives was supported by phenotypic, physiological, and biochemical analyses. Based on the combined genomic, phenotypic, and biochemical evidence, we propose the establishment of the novel rhizobial species Martinezella legmii sp. nov., with strain LEGMi-135b designated as the type strain (LEGMi-135b), and Martinezella ivanii sp. nov., with strain LEGMi-198b designated as the type strain (LEGMi-198b). This study expands our understanding of rhizobial diversity associated with nitrogen-fixing symbioses, particularly among tropical leguminous plants from Costa Rica

Antimicrobial resistance (AMR) is one of the greatest challenges within the One Health continuum. Exploring transmission routes between health domains and determining their driving forces are key priorities for future research. This effort can be effectively supported by landscape epidemiology, a field of science that integrates methods from landscape ecology and epidemiology to unravel the complex interdependencies behind disease transmission. This exploratory study aimed to demonstrate that landscape diversity and the degree of hemeroby (anthropogenic impact) correlate with the composition of bacterial communities and their AMR profiles. To test this hypothesis, submandibular lymph nodes from Cervidae and Suidae were collected to detect Staphylococcus and Mammaliicoccus bacteria and characterise their AMR features using selective culture and the VITEK 2 Compact automated system. As a result, the bacterial community in the more natural landscape was more diverse, characterised by the dominance of Mammaliicoccus sciuri and pan-susceptible isolates of Staphylococcus hyicus, and it displayed a low-level, heterogeneous AMR profile. Within the more hemerobic landscape, the bacterial community was characterised by the dominance of Staphylococcus epidermidis, a human-adapted species, and the AMR profile showed signs of higher antimicrobial pressure from both public health and veterinary origins. Although this study was based on only two study sites and was therefore not suitable for drawing definite conclusions, the findings suggest that human impact manifests itself in both bacterial and AMR profiles. A high prevalence of mammaliicocci and a heterogeneous AMR profile appeared to be indicators of naturalness. Conversely, the dominance of a human-adapted bacterial species and the accumulation of AMR features characteristic of medical environments likely indicate higher degrees of hemeroby.

Improving environmental management in the offshore Oil & Gas sector requires approaches that capture the ecosystem services (ES) provided by marine sediments, particularly their roles in carbon and nutrient cycling. Microbial communities are central to these processes, and molecular tools offer new opportunities to assess their functional diversity. To explore how different sequencing approaches inform environmental impact assessment, we compared taxonomic and functional prediction based on 16S metabarcoding, shotgun metagenomics and messenger RNA-based metatranscriptomics, targeting prokaryotic communities. Our aims were to evaluate the ability of each approach to detect impact and to determine how well they captured functions relevant to ES. All approaches revealed clear differences in community composition between impacted and non-impacted sediments at both taxonomic and functional levels, with impact significantly associated with hydrocarbon and barium content. Functional inventories showed substantial overlap across the three approaches, and 48-55 ES-related processes were detectable in all datasets. While metagenomics provided the strongest statistical discrimination between impact groups, metatranscriptomics resolved the actively expressed pathways underpinning ES, yielding the most biologically meaningful functional profiles despite its lower statistical power. All approaches indicated that Oil & Gas activity drives shift towards anaerobic, hydrocarbon-degrading, and sulfur-respiring microbial communities, with hydrocarbon degradation, sulfur cycling, and metal detoxification being the dominant ES processes in impacted sediments. Metabarcoding was confirmed as a cost-effective option for impact assessment when focused on taxonomic composition. However, functional prediction from metabarcoding data proved less reliable, as several ES showed contrasting associations to impact category between metabarcoding and shotgun sequencing approaches.

Soil nematodes play key roles in regulating organic matter decomposition, nutrient cycling and trophic interactions in the soil ecosystem, and are very useful bioindicators of soil condition and soil health. But to characterise them, we need reliable measurements of nematode biodiversity in soils. DNA-based techniques such as metabarcoding have recently explored their biodiversity, with successful results. However, there are many methodological steps that can modify the outcome considerably and require informed decisions. In this study, we tested the effect on nematode alpha and beta diversity metrics of three methodological steps, namely i) type of extraction of genetic material from the matrix, either from soil directly with different starting amount of material or extracting nematodes first via the Baermann or Oostenbrink methods, ii) the choice of taxonomically-relevant DNA segment for amplification, with a nematode specific primer (NemF/18Sr2b) and a universal eukaryotic primer (3NDf/1132rmod), both within 18S rDNA, and iii) the selection of appropriate reference library for taxonomic assignment (comparing three nematode specialised databases and three broad taxonomic databases). Observed differences in alpha diversity were more pronounced when comparing the nematode extraction vs soil extractions. In particular, direct soil extractions co-extracted many non-nematode taxa, especially if amplified with a universal primer, which can significantly reduce the number of nematode reads obtained and therefore the accuracy of nematode community indexes and other nematode-based metrics. The choice of the reference database strongly affected the taxonomic identification, which calls for extreme caution when making this selection. Notably, specialised databases tended to over assign reads to nematodes that were detected as non-nematodes by broad taxonomic databases. However, beta diversity metrics were consistent across methods, highlighting these metrics as robust against methodological choices. Our study clearly demonstrates that the choices of methods for nematode biodiversity studies using molecular tools can have a big impact on the results, and decisions should be made consciously and carefully.