Amplicon sequencing has long served as a robust method for characterising microbial communities, and despite inherent resolution limitations, it remains a preferred technique, offering cost‐ and time‐effective insights into bacterial compositions. Here, we introduce ONT‐AmpSeq, a user‐friendly pipeline designed for processing amplicon sequencing data generated from Oxford Nanopore Technology (ONT) devices. Our pipeline enables efficient creation of taxonomically annotated operational taxonomic unit (OTU) tables from ONT sequencing data, with the flexibility to multiplex amplicons on the same barcode. The pipeline encompasses six main steps—statistics, quality filtering, alignment, clustering, polishing, and taxonomic classification—integrating various state‐of‐the‐art software tools. We provide a detailed description of each step, along with performance tests and robustness evaluations using both test data and a ZymoBIOMICS^® Microbial Community Standard mock community dataset. Our results demonstrate the ability of ONT‐AmpSeq to effectively process ONT amplicon data, offering valuable insights into microbial community composition. Additionally, we discuss the influence of polishing tools on taxonomic insight and the impact of taxonomic annotation methods on the derived microbial composition. Overall, ONT‐AmpSeq represents a comprehensive solution for analysing ONT amplicon sequencing data, facilitating streamlined and reliable microbial community analysis. The pipeline, along with test data, is freely available for public use.

In European countries, raccoon dogs (Nyctereutes procyonoides) are believed to impact ecosystems and potentially threaten native species. In this study, the stomachs of 525 raccoon dogs culled in Danish wetlands were analysed and compared to 53 stomachs of adult raccoon dogs killed by vehicles, from the countryside. The stomach content was analysed using traditional macroscopic technics supplemented by eDNA analysis. The diet of raccoon dogs in wetlands did not differ notably from that of raccoon dogs in other parts of Denmark except for the larger amount of bait, and less shrews and fruit. More than 80% of the diet of adult raccoon dogs in wetlands consisted of bait, birds (mainly ducks taken in autumn and songbirds), amphibians, carrion, berries, fruit, and grass. Also, insects (mainly larger beetles and aquatic insects) occurred in the diet of raccoon dogs in wetlands. Waterfowl eaten during the autumn and winter are likely to be migrating birds. A high number of carrion beetles and fly maggots indicates a high consumption of carrion of all kinds. Eggs of ground-nesting birds made up a negligible part of the raccoon dog’s diet. eDNA-analysis found no trace of shorebirds and only common species of amphibians were recorded. This study provides no evidence that raccoon dogs are a threat to ground-nesting birds nor to endangered amphibians in Danish wetlands.

For the purpose of nature management and species conservation, European bison (Bison bonasus) are being increasingly reintroduced into nature reserves across Europe. The aim of this study was to investigate European bison’s adaptability to new areas through the study of their parasite-EPG (eggs per gram feces) and dietary diversity during twelve months after translocation. We compared the parasite-EPG from introduced European bison in Lille Vildmose, Denmark, with the parasite-EPG from populations from Bornholm, Denmark, and Białowieża Forest, Poland. From March 2021 to February 2022, fecal samples were collected from three populations. Samples from Lille Vildmose were examined through flotation, sedimentation, the Baermann technique, and nanopore sequencing. Fecal samples from Bornholm and Białowieża were examined through flotation and sedimentation. Nanopore sequencing of DNA from 63 European bison’s fecal samples collected during March–September in Lille Vildmose identified 8 species of nematodes within the digestive tract of the European bison, with Haemonchus contortus being the most frequently observed. In Lille Vildmose, a significantly higher excretion of nematode-EPG was observed during the summer period than in the spring, autumn, and winter. In addition, monthly differences in the excretion of nematode eggs were found, with this being significantly higher in June than in the months during autumn and winter (October–February). Significant differences in the nematode-EPG were only found between the excretion of nematode eggs in Białowieża Forest when compared to that of Lille Vildmose, with significantly higher excretion in Lille Vildmose (October–November). The results indicate that the development rates for nematodes may be affected by changes in temperature, with increasing temperatures speeding up their development time. Independent of this study design, wildlife vets together with the gamekeepers managing the herd found it necessary to treat the herd with antiparasitics for practical and animal welfare reasons in relation to translocation. Furthermore, 79 plant taxa were identified in the diet of the European bison. The broadest diet was observed in March suggesting that the European bison quickly adapted to their new habitat. The results suggest a seasonal shift in their diet, with this being most apparent from March to April.

Xanthine oxidase (XO) and lactoperoxidase (LPO) are highly abundant enzymes in milk. Their substrates, xanthine and thiocyanate, are found in elevated amounts in infant saliva, leading to a proposed interaction between milk and saliva referred to as the XO-LPO system. This system is suggested to generate reactive oxygen and nitrogen species with potential antibacterial effects. The antibacterial activity of the XO-LPO system was assessed on bacteria cultured from the oral cavities of five infants, where a reduction in bacterial growth rate was observed at 40 µg mL^-1 of each enzyme and with complete inhibition achieved at 200 µg mL^-1. Gene expression analysis showed that XO-LPO treatment led to downregulation of several reactive oxygen species-related genes, suggesting a transient bacterial stress response. The study also observed downregulation of key glycolytic enzymes, indicating that XO-LPO treatment affects bacterial metabolism at transcriptional level, suggesting a possible mechanism of action for the XO-LPO system. Collectively, these findings offer new insights into the XO-LPO system, revealing novel aspects of the interaction between lactation and microbiome influence.

Arctic regions are inhabited by terrestrial ectotherms that have adapted to an extreme environment where food resources are limited. The host associated microbiome may partly explain their ability to live under these conditions, but very little is known about the microbiome of Arctic ectotherms. We investigate how the bacterial community of the Greenlandic seed bug ( Nysius groenlandicus ) and damsel bug ( Nabis flavomarginatus ) is affected by different abiotic and biotic factors (time, acclimation temperature, humidity, and diet) under both field and laboratory conditions. We found large differences in the bacterial composition and diversity between the two species including species-specific presence of potentially symbiotic bacteria. The bacterial community of both species changed across the season, which may be explained by the changing climatic conditions, such as temperature and humidity. This was further supported by results from the laboratory experiments. We also found that diet changed the bacterial composition in both species and that bacteria could be transferred from prey to predator. Together, these results show that the bacterial community of some Arctic insects are highly dynamic and modulated by different abiotic and biotic factors, suggesting that the microbiome plays an important role for these organisms to persist in an extreme and resource-limited Arctic environment.

Diseases are an important but poorly understood influence on marine ecosystems. However, many pathogens of wild marine organisms remain poorly characterized, limiting our understanding of their ecology and potential impacts. This study investigates the potential virulence mechanisms of a Shewanella algae strain isolated from the brain tissue of a deceased northern bottlenose whale (Hyperoodon ampullatus). Using long-read Oxford Nanopore RNA Sequencing, differential gene expression analysis (DGEA) was conducted comparing growth in standard media, Tryptic Soy Broth (TSB) and TSB supplemented with 5% brain homogenate (BH) to stimulate expression of genes potentially associated with virulence. Twenty-eight significantly differentially expressed genes (23 identified using the assembled genome and five additional non-overlapping genes detected using a closely related reference genome) were identified out of 7,213 genes with at least two primary mapped reads. These genes were associated with processes that have been linked to bacterial virulence in previous studies, including protein translocation (e.g., small signal recognition particle), flagellar motility, chemotaxis, and iron acquisition. Functional analysis indicated an increased expression of pathways related to amino acid metabolism and motility, whereas carbohydrate and inorganic ion transport were downregulated. These results suggest that environmental cues, potentially including factors related to iron metabolism, may influence bacterial virulence, obtained using Oxford Nanopore RNA sequencing for pathogen transcriptomics.