Environmental DNA

O_LIEnvironmental DNA (eDNA) metabarcoding is an emerging tool for biodiversity assessment in freshwater systems, offering high-resolution insights into community composition. Here, we apply eDNA metabarcoding to evaluate the ecological impacts of Eurasian beaver (Castor fiber) activity within a seminatural enclosure in the Scottish Highlands. C_LIO_LIWe collected seasonal water samples from nine sites, six influenced by beaver dams and three control sites with no evidence of beaver engineering, across a 40-hectare enclosure. Samples were analysed for vertebrate and macroinvertebrate diversity using established 12S and COI markers. C_LIO_LIVertebrate alpha diversity did not differ significantly between beaver and control sites, likely reflecting the small spatial scale and low species richness of upland Scottish streams. However, community composition differed significantly between treatments, especially for fish (PERMANOVA, R2 = 0.55, P < 0.001), with beaver-influenced sites dominated by three-spined stickleback and control sites by brown trout. Macroinvertebrate communities showed a 78% increase in gamma diversity in beaver-modified habitats relative to controls. Species composition varied strongly with beaver presence (PERMANOVA, R2 = 0.29, P < 0.001), likely due to the creation of lentic-lotic mosaics and associated microhabitat diversity. Seasonal variation was significant in both taxonomic groups, with the lowest species richness and highest community dispersion observed in summer, probably reflecting hydrological and temperature-driven dynamics in eDNA production and transport. C_LIO_LIOur findings reinforce previous evidence that beaver dam-building activity enhances beta diversity in headwater systems. Additionally, we demonstrate that eDNA metabarcoding is a sensitive method for detecting spatial patterns in freshwater biodiversity associated with these activities at scales ranging from tens to hundreds of meters. These approaches could inform future monitoring strategies aligned with landscape-scale beaver management and reintroductions. C_LI

Terrestrial environmental DNA (eDNA) approaches are rapidly expanding, yet robust, field-ready substrates for detecting insect DNA remain limited in forest ecosystems. Tree sap is a localized microhabitat that attracts diverse insects and may provide a useful substrate for surface eDNA sampling, but its potential for insect monitoring has rarely been evaluated. Here, we present a pilot proof-of-concept study testing naturally exuding tree sap and sap-mimicking traps as terrestrial eDNA substrates. We collected swab samples from sap and trap surfaces at two forest sites in Japan (Fujisawa and Minamisanriku) and performed metabarcoding using COI and an arthropod-focused 16S marker (gInsect). Reads were processed into amplicon sequence variants and assigned by BLAST top hits against NCBI nt, with high-confidence detections defined at identity [&ge;]98%. Across sites, sap and trap swabs yielded multiple high-confidence insect detections spanning several orders, including sap-associated stag beetles (Dorcus spp.). Overlap with contemporaneous conventional monitoring was limited, suggesting that sap-surface eDNA and conventional surveys capture partly different components of sap-associated insect assemblages. In a targeted 2024 spot survey, actively fermenting sap yielded multiple insect eDNA detections, whereas inactive, non-fermented sap yielded no high-confidence insect detections. Although limited by small sample size and the absence of dedicated process controls, these findings support the feasibility of tree sap as a localized terrestrial eDNA substrate and provide a basis for future replicated studies of sap-associated insect monitoring.

Evidence supporting the use of airborne eDNA for biodiversity studies and ecosystem monitoring is growing. The promise of wide-area population dynamics data for downstream applications in targeted monitoring of pests and pathogens for agriculture and rare species for conservation is appealing; however, several technical challenges persist. Here, we focused on the development of a comprehensive dataset to facilitate assay development and accelerate the use of aerial sampling for species detection. Year-round metabarcoding data was generated using bacterial, fungal, plant, and arthropod primer sets and resulted in relative abundance estimates for 4,960 amplicon sequence variants (ASVs), 1,748 ASVs of which were assigned to a minimum taxonomic level of genus (bacteria, fungi, plants) or class (arthropods). Sequence diversity assessments and seasonal clustering based on presence/absence detection patterns were performed for individual ASVs, while discerning quantitative changes in seasonal abundance required grouping ASVs to at least the genus level. Examination of the technical aspects of metabarcoding suggested that the use of subsampling allows for consistent detection of genera with relative abundance values above 2 %, even when samples have varying sequencing depths. Sequencing depth was the primary determinate for detecting sporadic and/or rare ASVs. Sampler comparisons, common sources of variation, and the benefits of barcoding regional species to supplement the existing taxonomic databases were discussed. Insufficient knowledge of sampler coverage area for the different organism types was identified as a limitation to the deployment of aerial monitoring networks. Considerations for further aerial metabarcoding efforts are suggested based on our experimental findings. ImportanceOur study deals directly with the generation, analysis and limitations of airborne eDNA metabarcoding data for re-use by the broader environmental research community. This includes timing of seasonal detection for possible genera of interest across multiple kingdoms, including bacteria, fungi, plants and animals (specifically arthropods), and support for the generation of local databases to assess the current limitations of universal primers for species/genus taxonomic resolution. With regards to methodology, it continues to build upon established best practices for airborne eDNA collection in areas such as sub-sampling and sampling replicates, sampler type and sequencing depth. To accelerate possible uptake and application of the data, we provide the identified ASVs and their seasonal relative abundances as a resource.

Early detection and monitoring of non-indigenous species (NIS) is crucial to prevent their establishment and to reduce ecological and economic impacts in coastal ecosystems. Traditional monitoring approaches, which rely largely on morphological identification of collected organisms, are often time-consuming and may fail to detect species that occur at low abundance, are morphologically cryptic, or are present in the form of inconspicuous life stages. DNA-based approaches, particularly those resorting to environmental DNA, have demonstrated high aptitude for biodiversity monitoring and biosecurity surveillance. By examining the genetic material from bulk community samples or released into the environment, DNA-based approaches enable the detection of species without the need for direct observation, thereby increasing detection sensitivity and expanding the scope of monitoring programs. Despite the rapid growth of its employment in marine monitoring, a global synthesis of the status and trends of DNA-based approaches for detecting NIS in this environment has been lacking. Here, we present such synthesis, based on 146 published studies employing DNA for NIS detections in coastal environments. Two main methodological approaches were used across the reviewed studies, namely DNA metabarcoding which was applied in 49% of studies, closely followed by targeted single-species PCR assays, used in 42% of the studies. A smaller proportion of studies (10%) combined both approaches, integrating broad community screening with targeted detection to improve surveillance efficiency. Globally, 752 NIS were detected across disparate taxonomic groups, with metazoans representing the largest proportion of detections (464 species), followed by Chromista (210 species) and Plantae (77 species). Among these, the most frequently detected taxonomic groups included Dinophyceae (Dinoflagellata), Teleostei (Chordata), Florideophyceae (Rodophyta), Polychaeta (Annelida), Copepoda and Malacostraca (Arthropoda), and Ascidiacea (Chordata). At the species level, several well-known marine invaders were recurrently reported, including Bugula neritina (Linnaeus, 1758), Styela plicata (Lesueur, 1823), Acartia (Acanthacartia) tonsa Dana, 1849-1852, and Botryllus schlosseri (Pallas, 1766), highlighting the ability of DNA approaches to detect widespread and established invaders across different regions. The mitochondrial cytochrome c oxidase subunit I (COI) gene was the most widely used genetic marker, reflecting its broad taxonomic coverage and extensive representation in reference databases, particularly for targeting Metazoa. Ribosomal RNA genes, particularly 18S and 16S rRNA gene markers, were also frequently employed to target a wider range of eukaryotic taxa. Regarding sampled substrates, water was by far the most analyzed substrate, followed by zooplankton and biofouling communities collected from man-made structures. Notably, approximately 31% of all NIS detections reported in the reviewed studies constituted new regional records. These results highlight the potential of eDNA for coastal monitoring but also underline important limitations. Persistent geographical, taxonomic, and methodological biases can affect detection outcomes, and reliance on single sample types or markers may increase false negatives - particularly critical for NIS early detection. Therefore, multi-marker and multi-substrate approaches are essential to improve detection reliability and support effective biosecurity strategies. As reference databases continue to expand and methodological protocols become increasingly standardized, DNA-based monitoring is likely to play a central role in future management and surveillance of biological invasions in coastal ecosystems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/722998v1_ufig1.gif" ALT="Figure 1"> View larger version (75K): org.highwire.dtl.DTLVardef@17948b1org.highwire.dtl.DTLVardef@193832dorg.highwire.dtl.DTLVardef@189033dorg.highwire.dtl.DTLVardef@33cddf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Understanding risks of biological invasions associated with ballast water (BW) requires full understanding of the biodiversity transported in ballast tanks. Here we characterize the remarkable level of diversity that can be carried in the BW of a single vessel. To maximize our ability to capture BW diversity we: 1) utilized DNA-based methods to describe biodiversity, including both native and non-native taxa; 2) exploited multiple primer sets targeting multiple genomic loci with different expectations for taxonomic coverage; 3) assessed multiple tanks on a single vessel to capture different communities present in different tanks; and 4) sampled those tanks with far higher-than-usual replication both to improve representation of the diversity present and to enable statistical estimation of total richness. Using this approach, we found extraordinarily high diversity associated with a single vessel. Across all loci, we estimate a total of 272 taxa that can be assigned species names; looking more broadly at unnamed molecular operational taxonomic units, our estimates are between 425 and 742 individual taxa, depending on the locus. We confirm that only a fraction of this diversity would be captured with typical sampling efforts. We found that different loci capture different snapshots of biodiversity and that our ability to detect taxa of interest (e.g., non-native species) depends on sampling effort and genomic locus. Our results expand upon previous studies describing highly diverse BW communities and add to a growing literature that demonstrates the value of molecular methods for characterizing those communities and assessing the associated risk of non-native species introduction.

Metabarcoding is a powerful tool for biodiversity comparisons, where standard-size DNA barcodes (>500 bases) offer better taxonomic resolution than shorter ones. Still, the choice of sequencing platforms and bioinformatics pipelines may strongly affect inferred diversity due to various technical biases. We assessed the relative performance of Illumina MiSeq i100 (2x500 paired-end), PacBio Revio and Oxford Nanopore MinION sequencing and bioinformatics pipelines, using full-length ITS amplicon sequencing datasets from a 103-species mock community and 45 composite soil samples. Despite numerous low-quality reads, PacBio yielded the lowest overall error rate and highest number of taxa. Illumina revealed the highest proportion of chimeric and index-switched reads, along with a strong bias towards shorter amplicons. MinION data analysed using PRONAME and Minovar - a bioinformatics pipeline presented here - had the largest proportion of low-quality data, and rare taxa were lost during data filtering and read polishing steps. Although Minovar enabled amplicon sequence variant (ASV) level precision for common taxa, we recommend clustering ASVs into OTUs. For PacBio, standard filtering approaches outperformed the ASV approach because they retained rare taxa. For Illumina, a stringent ASV approach or removal of rare OTUs would limit artefacts. Across all platforms, excess PCR cycles promoted chimeric and low-quality reads and lost quantitativity in biodiversity assessments. With moderate differences in effect sizes, all analytical approaches supported the conclusion that sampling design determines how we see soil biodiversity responses to land use. For biodiversity surveys based on the full-length ITS metabarcoding, we recommend using PacBio sequencing with standard, non-ASV pipelines.

The marbled teal (Marmaronetta angustirostris) is a widely distributed but declining waterfowl species, classified as Near Threatened globally and Critically Endangered in Spain. Despite ongoing conservation actions, including ex situ management and population reinforcement programmes, the genomic consequences of long-term captivity, inbreeding, and patterns of functional genetic variation remain unknown due to the absence of a species-specific reference genome. Here, we present the first chromosome-level genome assembly for this species. The genome was generated using PacBio HiFi long reads and Omni-C data, yielding a 1.15Gb assembly with a scaffold N50 of 76.95Mb. A total of 97.16% of the assembly was anchored into 36 chromosome-scale scaffolds, including the Z and W sex chromosomes. BUSCO analysis recovered 99.2% of conserved avian genes. Gene prediction was performed using both ab initio and homology-based strategies, resulting in 16,048 protein-coding genes. This resource provides a foundation for genomewide analyses of inbreeding, demographic history, and adaptive variation, and will support evidencebased in situ and ex situ conservation strategies for this threatened species.

The sunflower sea star (Pycnopodia helianthoides) suffered a catastrophic population decline across its range from 2013 to 2017 due to the devastating Vibrio pectenicida FHCF-3 driven sea star wasting disease (SSWD) pandemic with minimal signs of population recovery. The functional extinction of this apex predator across substantial parts of its range has created a need to identify and track the remaining intact populations. Environmental DNA (eDNA) approaches provide a simple, cost-effective, and non-destructive method for monitoring occurrences, and in some cases abundances, of marine species, consistently outperforming visual occurrence monitoring efforts in sensitivity, speed, and cost. Here, we designed, developed, and validated a P. helianthoides-specific eDNA assay to identify refugia, using both quantitative and digital droplet PCR approaches. We first generated the most comprehensive sea star mitochondrial genome reference database to date (n=93 taxa, n= 15 novel). We then used unikseq and Geneious bioinformatics software to identify the unique nad5 gene region and design a highly specific hydrolysis probe-based PCR assay. We validated the performance of this assay through laboratory, mesocosm, and field testing, demonstrating a highly specific and sensitive assay. In a field application of the new assay across regions in British Columbia, Canada, we found a positive correlation between P. helianthoides eDNA concentrations and biomass density, especially when appropriately accounting for spatiotemporal integration scales (R2=0.67). The eDNA assay provides a rapid and scalable tool for monitoring the sunflower sea star which has been proposed for listing as threatened under the U.S. Endangered Species Act of 1973. Molecular tools like the one presented here enhance management and recovery efforts not only by identification and monitoring of remnant wild populations, but also by helping to assess population level response and recovery following reintroduction efforts.

Environmental DNA (eDNA) surveys are increasingly used to assess marine biodiversity and inform deep-sea environmental decision-making, including mineral resource management and fisheries oversight. Yet standard low-volume protocols inherited from coastal work may be inadequate at depth, and no quantitative framework links depth and ecosystem context to defensible filtration volume targets. We compiled 841 eDNA samples from eight expeditions across the North Atlantic, Wider Caribbean, and Pacific (surface to 4000 m) to quantify how recoverable eDNA scales with depth and surface productivity, and to derive depth- and productivity-aware sampling targets. Total eDNA concentration declined with depth as a power law, with attenuation exponents (b) modulated by surface productivity: most gradual in eutrophic waters (b = 0.67), intermediate in mesotrophic (b = 0.90), and steepest in oligotrophic systems (b = 1.25); volume-weighted models explained 66-88% of the variance. At a fixed extract-concentration target, required filtration volumes diverged ~7-fold between oligotrophic and eutrophic systems at 200 m and ~38-fold at 4000 m. Conventional Niskin sampling, therefore, undersamples deep-sea biodiversity, particularly in mid- to low-productivity systems. Among laboratory parameters, the assay-specific extract-concentration target exerted greater leverage on required volume than extraction efficiency or elution volume. Volume-aware sampling paired with optimized recovery should be routine in deep-sea eDNA surveys.

Global changes in land use and nutrient cycling are transforming ecosystems at unprecedented rates, with significant consequences for infectious disease dynamics. Aquatic environments are particularly vulnerable because the interplay of habitat modification, nutrient enrichment, and biodiversity loss can drive pronounced changes in the community composition of food webs, including hosts and parasites. Yet, despite well-documented effects of habitat modification on aquatic communities and food webs, the mechanisms through which these changes influence infectious disease dynamics remain poorly resolved. This gap arises, in part, because it remains challenging to disentangle how multiple stressors interact to shape disease outcomes and quantify parasite levels and host densities from field-collected samples. Here, we illustrate two tools that might help address these challenges. First, highly sensitive droplet digital PCR can quantify infection loads even when the signal:noise ratio is low. Second, stepwise Bayesian path analyses can identify the direct and indirect pathways connecting land-use changes to infectious disease dynamics. As a case study, we examined cyclopoid copepods and their helminth parasite, Schistocephalus solidus, across 47 freshwater lakes on Vancouver Island, a region strongly shaped by commercial logging, including widespread clear-cutting of old-growth forests. Our results reveal a positive correlation between copepod density and deforestation, potentially mediated by associated changes in water quality and calanoid copepods, key competitors of the focal host. ddPCR enabled sensitive detection of extremely low parasite signals in field-collected copepods. We detected positive infections in only 19.5% of the lakes surveyed, highlighting the difficulty of assessing disease dynamics in natural populations. Nonetheless, this study highlights the challenges of linking land-use change to disease outcomes, while also demonstrating that sensitive molecular and statistical tools offer new ways to reveal these hidden connections.

Near-shore coral reefs in southern Guam (Mariana Islands) experience severe sedimentation, in particular during the wet season when rainfall and erosion are high. We sampled fragments of the reef-forming coral Porites lobata from opposite ends of a sedimentation gradient in Fouha Bay, southern Guam, during dry and wet seasons. Using DNA metabarcoding, we characterized the diversity and composition of P. lobata-associated Symbiodiniaceae and bacterial microbiome communities. As in many species of Porites, Symbiodiniaceae communities of P. lobata were dominated by variants of Cladocopium C15 with sites showing differences in Symbiodiniaceae communities attributable to variation in these Cladocopium C15 variants. Bacterial microbiomes of P. lobata were dominated by Endozoicomonadaceae, a family of putative coral bacterial endosymbionts involved in nutrient cycling. Site and seasonal differences in bacterial diversity and community composition were apparent. In close proximity to the mouth of the river draining into Fouha Bay, bacterial diversity was highest during the wet season when sedimentation is generally severe. Microbiome reorganization in response to sedimentation may explain this result, but we also found overrepresentation of bacteria associated with terrestrial origin close to the river mouth and/or during the wet season. Together these patterns highlight that coral Symbiodiniaceae and bacterial communities are both spatially and temporally structured in this disturbed system. IMPORTANCEThis study provides a time series dataset of coral-associated microorganisms, including dinoflagellate algae and bacteria, from a tropical bay impacted by sedimentation that results from upstream erosion of disturbed soils. Characterizing temporal patterns of coral-associated microbes provides insights into the dynamic nature of these communities. While microbiome variability across sites and seasons may be a result of acclimatization to different environmental conditions, we identified bacterial groups of putative terrestrial origin in sampled coral microbiomes that may have been exported from eroded soils to the near-shore reef. Considering that disturbed soils act as hotspots for the proliferation of potentially harmful substances, such as antimicrobial resistance genes, understanding microbial community connections at the marine-freshwater-terrestrial interface is an important step toward evaluating environmental impacts across connected ecosystems from ridge to reef.

Telomere length (TL) is increasingly used in ecology as a biomarker of individual quality and environmental stress, yet research on non-model species with complex life histories remains limited. Because TL varies among tissues and across ages in a species-specific manner, identifying non-lethal tissues that reliably reflect whole-organism telomere dynamics is essential for longitudinal telomere studies in the field. This study aimed to evaluate tissue-specific TL in Japanese eel (Anguilla japonica), an endangered catadromous fish. We first mapped the chromosomal distribution of telomeric sequences using fluorescent in situ hybridization (FISH), the first application of this method in this species. We then tested whether muscle and caudal fin, which can be sampled easily and non-lethally, can serve as suitable proxy tissues for TL measurements in wild individuals. Relative telomere length (RTL) was quantified by qPCR in blood, brain, caudal fin, gonads, heart, liver, and muscle. FISH analysis confirmed telomeric repeats at all chromosomal ends, with only weak interstitial signals on three chromosomal pairs unlikely to affect qPCR-based estimates. A generalized additive mixed model and Wilcoxons signed-rank tests revealed significant inter-tissue differences: RTL was shortest in the brain and muscle and longest in liver, blood and caudal fin. Muscle and caudal fin RTL were significantly correlated with RTL in many other tissues, supporting their use as proxy tissues for longitudinal TL monitoring, including responses to environmental variation. Both total length and age were tested as explanatory variables for RTL, and the model including total length showed a better fit than the age-based model. Non-linear relationships between RTL and total length observed in several tissues suggest physiological shifts associated with growth and sexual differentiation. Overall, these findings advance understanding of telomere dynamics in eels and establish muscle and caudal fin as suitable tissues for repeated, non-lethal TL assessment in ecological and conservation contexts.

Philippine freshwater ecosystems are considered one of the most diverse ecosystems harboring numerous fish species. However, in the Philippines, these ecosystems are threatened by invasive species that potentially disrupt ecological balance. In this study, we focused on the vermiculated sailfin catfish Pterygoplichthys disjunctivus, an invasive aquarium species reported in several Philippine aquatic ecosystems. Despite its documented spread, its potential range under a rapidly changing climate remains poorly understood for the country. Hence, in this study, we utilized the MaxEnt model to predict its near-current and future habitat suitability in the Philippines. Using 11 reported occurrences, our model showed high predictive accuracy (AUC = 0.882{+/-} .034, TSS = 0.7394 {+/-} 0.154, SEDI = 0.971 {+/-} 0.019). Across the current and future scenarios, slope was the primary contributor (78.7% - 81.3%), followed by BIO 10 or the mean temperature of the warmest quarter(18% - 27.8%), and flow accumulation (0% - 5.2%). However, for the SSP126 scenario, BIO10 is projected to triple by 2050 (18 - 48%). Current projections identify high-risk regions, particularly central Luzon (Laguna de Bay and Lake Taal), the Cagayan River Valley, and portions of eastern Mindanao (Agusan Marsh and Lake Mainit). Sankey transition analysis confirms a high habitat stability rate (>73%) for high-suitability pixels in both SSPs, indicating persistent invasion risk. Overall, our study provides a framework for invasive species management and contributes to the conservation of Philippine aquatic ecosystems.

Antimicrobial resistance (AMR) genes are increasingly recognized as an emerging environmental contaminant. Yet, the ecological mechanisms shaping their distribution across natural landscapes remain poorly understood. Here, we quantified AMR gene abundances in microbial communities sampled from wild fish from eight freshwater lakes on Vancouver Island and paired these gene-level measurements with fine-scale limnological and land-use data. Using droplet digital PCR, field surveys, and an iterative spatial forecasting framework that integrates Random Forest models with regression kriging, we explored how watershed-scale processes relate to variation in AMR genes across lakes. Our analyses reveal potential associations between elevated AMR gene levels, changes in water quality, deforestation, and geographic proximity to salmon aquaculture. By integrating data across biological and spatial scales, from genes within microbial communities to lake-level conditions and landscape patterns, this study illustrates the value of combining quantitative molecular measurements with geospatial modeling to identify environmental factors that may promote antimicrobial resistance in natural systems. Our approach provides a proof-of-concept and a general predictive framework for generating hypotheses and informing future monitoring efforts aimed at understanding, managing, and forecasting environmental reservoirs of resistance. SignificanceAntimicrobial resistance (AMR) genes are ancient components of environmental microbiomes. Yet, the mechanisms that generate modern hotspots of resistance across natural landscapes remain unclear. Here, we reveal how watershed-scale environmental change, including water quality metrics linked with deforestation and proximity to salmon aquaculture, predicts elevated AMR gene levels in the microbiomes of wild fish populations. By combining quantitative droplet digital PCR with ecological data and geospatial modeling, we move beyond isolated surveillance data to identify ecological mechanisms that promote antimicrobial resistance in freshwater ecosystems. This integrative approach provides mechanistic insight into why certain habitats, and the organisms within them, become reservoirs of resistance while others do not. Our findings highlight the importance of ecological context in understanding resistance evolution and offer a predictive tool for informing proactive monitoring and management strategies.

Seafood nutrients from global fisheries are of increasing importance for research and policy in food security and nutrition. As the chemical composition of fish is determined by what they eat, their energetic demands, and the environment in which they live, nutrient content reflects aspects of physiology and life history, ecological and environmental traits, as well as evolutionary history. Here we present data from Bayesian model estimates of 12 key nutrients (calcium, iron, phosphorus, magnesium, selenium, zinc, vitamin A, vitamin B9, vitamin B12, vitamin D, omega-3 fatty acids, and protein) in wild fish, using a database of reported nutrient content for freshwater and marine species. We then predict the nutrient content of 5588 fish species with traits available from FishBase. We compare our previous model using traits alone with a new model of both traits and phylogeny, and present the data, code, and predictions for models coded in PyMC. These models and predictions, made freely available through FishBase, can be used to explore the historical, current, and future nutrient content of fisheries catch.

Ocean warming is altering abiotic environments and biotic interactions experienced by marine organisms, where sensitive early developmental windows occur in biologically complex seawater communities. The impact of these interactions on developmental processes and fitness in hosts is not well understood, but likely contingent on the establishment of a host-associated microbiome. Here, we hypothesize that temperature and microbial exposure during embryogenesis influence larval microbiome assembly and host morphology. Strongylocentrotus purpuratus embryos were raised in low microbial richness (LMR) or high microbial richness (HMR) seawater at ambient (14 {degrees}C) or elevated (18 {degrees}C) temperature, then collected at 2, 4, and 6 days post-fertilization (dpf) following multiple feedings. Higher microbial diversity was observed in larvae that developed in HMR seawater when compared to LMR. Differences in relative abundances of dominant microbial families between seawater and larvae suggest some degree of host selectivity in microbiome assembly. Temperature did not strongly alter microbiome composition, but both temperature and microbial condition led to differences in larval morphology by 6 dpf, potentially due to enrichment of microbes with chemoheterotrophic functions. By linking how temperature and microbial communities interact with host development, we contribute novel insights into how early-life environmental conditions impact holobiont formation and morphology. One sentence summaryEarly developmental temperature and microbial conditions shape larval microbiome establishment and morphology.

Many pathogens, both those with human spillover potential as well as avian-specific viruses, are maintained in wild bird populations. While routine surveillance for influenza A viruses (IAVs) is performed annually, surveillance for other pathogens is limited. Sampling of wild birds is time-consuming, labour-intensive, often limited in sample size, and involves handling of wild and potentially infected birds, posing an increased risk of direct exposure for personnel. Additional methods for surveillance are needed given these significant challenges. Longitudinal fecal and sediment sampling was performed at various sites in southern Manitoba, Canada, particularly focused in Winnipeg from May to October 2025. Sites were chosen based on the suitability of the area for waterfowl habitat, the presence of waterfowl in the area, as well as proximity to reported outbreaks of H5N1 influenza virus. Fecal and sediment samples were collected and screened for the presence of influenza A virus (IAV), Newcastle disease virus (NDV), avian reovirus (ARV), and avian poxvirus (APXV). In total, 782 combined fecal and sediment samples were collected. Of the 714 fecal samples, 34 tested positive for IAV RNA (4.8% prevalence). None of the IAV-positive fecal samples tested positive for H5 RNA. Of the 68 sediments, 15 were positive for IAV RNA (22.1% prevalence), four of which were positive for H5 RNA. NDV RNA positivity was low, with only four positive fecal samples (0.6% prevalence) that were all collected on the same day. ARV RNA positivity was also low, with five positive sediment samples (7.4% prevalence in sediment samples). None of the samples tested positive for APXV DNA. This study builds on previous work showing the utility of environmental sampling for a variety of avian and zoonotic pathogens using a One Health approach that is low-risk, efficient, and high-throughput.

Bacteria require rapid adaptation under fluctuating environmental conditions. Commonly recognized global regulators enable bacteria to respond promptly to external changes, though they are either restricted to specific bacterial taxonomies or physiological statuses, suggesting that additional regulators are required for adaptation. DNA methylation is a reversible modification affecting bacterial gene regulation. However, conventional methods can only detect one DNA methylation form each round, leaving the understanding of DNA methylation in bacterial adaptation mostly unknown. This study aimed to identify genome-wide DNA methylation variation (N6-methyladenine, N4-methylcytosine, and 5-methylcytosine) in Escherichia coli under different culture conditions using Oxford Nanopore sequencing. DNA samples from six conditions (normal, low oxygen, low pH, high temperature, high salt, and recovery after low pH exposure) during the exponential and stationary phases were extracted. When culture conditions were compared to the normal condition, E. coli exhibited more differentially methylated sites during the exponential phase than in the stationary phase. During the exponential phase, the genes differentially methylated in all conditions were involved in cellular activities, such as cellular and metabolic processes. During the stationary phase, universally differentially methylated genes were associated with oxidation responses. Subsequent analysis found that although DNA methylation analysis was affected by batch effects, some genes (e.g. rpoS) showed consistently differential methylation across datasets. Our findings suggest that the E. coli DNA methylation profile was affected by growth phases and conditions, and DNA methylation profiling by Oxford Nanopore sequencing could be a potential approach for gene activity estimation in environmental samples. ImportanceBacterial DNA methylation is a reversible genetic modification affecting gene regulation, enabling rapid adaptation. Three major forms in bacteria are N6-methyladenine, N4-methylcytosine, and 5-methylcytosine. Using Oxford Nanopore sequencing, we characterized genome-wide variation in these methylation types in Escherichia coli under six conditions (normal, low oxygen, low pH, high temperature, high salt, and recovery after low pH exposure). DNA methylation signatures in E. coli varied with growth conditions. Using the normal condition as a baseline, E. coli during the exponential phase exhibited more differentially methylated genomic loci under stress conditions compared to the stationary phase. Under stress conditions, genes with differential methylation were associated with cellular processes or oxidative responses, depending on the growth phase. Our findings reveal that the DNA methylation signature in E. coli was affected by growth phases and conditions, and Oxford Nanopore-based DNA methylation profiling could be a potential approach for gene activity estimation in environmental samples.

The persistence of specialised survival spores produced by microbial pathogens represents a primary bottleneck in the management of plant diseases. In oomycetes, these spores (known as oospores) are largely impervious to chemical control, allowing them to persist in soil and initiate new infection cycles over many years. A prominent example is the soil-borne pathogen Phytophthora agathidicida, the causal agent of kauri dieback disease, where long-lived oospores hinder conservation efforts in native forests. The resilience of oospores is attributed to their thick wall composed of complex {beta}-glucan layers that render the oospores impermeable to most conventional biocides. Here we have investigated an enzyme-based approach for weakening the oospore cell wall. We searched enzyme databases to select {beta}-glucanases targeting a variety of linkages found in Phytophthora oospore walls. Eight of these {beta}-glucanases were successfully purified and tested for their digestive activity against intact oospores in vitro using a phenol-sulfuric acid assay. We showed that combining these enzymes was crucial to achieve significant digestion through synergies and additive effects. The optimal combination, comprising 1,3-, 1,6-, and 1,3(4)-{beta}-glucanases, was evaluated for its ability to permeabilise oospores to five biocides typically effective only on other, more sensitive lifecycle stages of the pathogen. Using a live/dead fluorescence assay, we observed that the effects of the membrane-targeting biocides were potentiated in oospores that were pre-treated with the {beta}-glucanase mixture. Our results highlight enzymatic cell wall permeabilisation as a promising strategy toward improved management of oospore persistence in kauri forest soils and against broader oomycete threats. KeypointsO_LIOur phenol-sulfuric acid assay can be used to screen for oospore-degrading enzymes. C_LIO_LISynergistic enzyme combinations are essential for effective oospore wall digestion. C_LIO_LIEnzyme pre-treatment sensitises oospores to membrane-targeting biocides. C_LI

Chelating ion exchange resins are widely used to eliminate metal interferences in the analysis of ammonium (NH4+) in soil extraction solutions. However, their potential to co-adsorb NH4+ remains underexplored. Here, synthetic metal ion solutions containing 6-30 mg L-1 NH4+ and the metal cations Ca2+, Mg2+, Cu2+, Mn2+, and Zn2+ were treated with Amberlite IRC-748 resin. The resin efficiently removed Ca2+ (-42.2%), Mg2+ (-21.1%), Cu2+ (-99.9%), Mn2+ (-56.9%), and Zn2+ (-93.6%). However, NH4+ losses of 2.2-5.6% were observed, indicating concentration-dependent co-adsorption. While these losses may be acceptable for concentration measurements via routine assays such as photometric analysis, they may still affect the accuracy of high-precision N analyses that rely on quantitative NH4+ recovery. This highlights a methodological caveat for resin-treated samples, especially in low-NH4+ environments. We therefore recommend including recovery assessments and correction factors when using chelating resins to improve accuracy in NH4+ quantification.