Sutton, W. | Search bioRxiv

bioRxiv2025

Effects of environmental setting and diet on the gut microbial ecology of eastern hellbenders (Cryptobranchus alleganiensis alleganiensis)

BackgroundEastern hellbenders (Cryptobranchus alleganiensis alleganiensis) have undergone substantial population declines throughout their range, leading them to become the focus of increased conservation efforts, including care in zoo and university settings. However, effective implementation of such conservation strategies often relies on a comprehensive understanding of host health, which can be directly influenced by the gut microbiome, yet characterization of gut microbiota often remains overlooked in ex situ conservation facilities. Additionally, effects on the gut microbiome associated with releasing zoo-reared animals into the wild are poorly understood. Therefore, these circumstances make hellbenders an ideal species to examine the relationship between zoo management strategies and gut microbial dynamics. Methods16S rRNA sequencing was used to investigate dissimilarities between the gut microbiome of hellbenders in zoo and wild settings and to evaluate the impact of implementing a wild diet in zoo-reared hellbenders. Additionally, the bacterial composition of zoo-released individuals and wild resident hellbenders was compared to examine the response of the gut microbiome upon release into natural habitat. Selected samples were also chosen for ITS1 rDNA sequencing as a preliminary investigation of the hellbender gut mycobiome. ResultsHuman rearing strongly affected the gut microbiome, leading to reduced bacterial richness as well as differing community structure than wild hellbenders. However, implementation of a wild diet in a zoo setting modulated the microbiome and appeared to be mainly driven by bacterial turnover. Additionally, both bacterial and fungal gut assemblages demonstrated the capacity for restructuring upon release into native habitat to become more reflective of a wild-type microbiome. ConclusionsWe completed the first study elucidating the gut microbial composition patterns of hellbenders, across both zoo and wild settings. These results provide an understanding of the potential impacts of conservation populations in zoos on gut microbial communities and also inform headstart programs of the transition of the gut microbiome post-reintroduction to the wild.

microbiology↗

bioRxiv2025

A comparison of short- and long-read whole genome sequencing for microbial pathogen epidemiology

Whole genome sequencing provides the highest resolution for characterizing pathogen evolution, epidemiology, and diagnostics. Genome assemblies contain information on the identity and potential phenotypes of a pathogen. Likewise, variant calling can inform on transmission patterns and evolutionary relationships. Recent improvements in Oxford Nanopore long-read sequencing have made its use attractive for genomic epidemiology. However, the accuracy and optimal strategy for analysis of Nanopore reads remains to be determined. We compared the use of Illumina short reads and Oxford Nanopore long reads for genome assembly and variant calling of phytopathogenic bacteria. We generated short- and long-read datasets for diverse phytopathogenic Agrobacterium strains. We then analyzed these data using multiple pipelines designed for either short or long reads and compared the results. We found that assemblies made from long reads were more complete than those made from short-read data and contained few sequence errors. Variant calling pipelines differed in their ability to accurately call variants and infer genotypes from long reads. Results suggest that computationally fragmenting long reads can improve the accuracy of variant calling in population-level studies. Using fragmented long reads, pipelines designed for short reads were more accurate at recovering genotypes than pipelines designed for long reads. Further, short- and long-read datasets can be analyzed together with the same pipelines. These findings show that Oxford Nanopore sequencing is accurate and can be sufficient for microbial pathogen genomics and epidemiology. Ultimately, this enhances the ability of researchers and clinicians to understand and mitigate the spread of pathogens. ImportanceGenome assembly and variant calling are important steps in microbial population studies and epidemiology. Most variant calling and genotyping pipelines are designed for Illumina short sequencing reads. Oxford Nanopore Technology long-read sequencing results in more complete genome assemblies but has historically been of lower quality. Here, we show that Nanopore long reads are now of sufficient quality for bacterial whole genome assembly and epidemiology. We benchmarked the accuracy of multiple variant-calling pipelines with short and long reads. Using an optimized variant calling approach, variant calls and genotypes inferred from long reads are as accurate as those inferred from short reads. Importantly, we found that gold-standard variant calling pipelines designed for short reads are also accurate with long reads when long reads are first fragmented into shorter sequences. This finding allows researchers to incorporate the advantages of Nanopore sequencing for genome assembly, while maintaining high accuracy for epidemiology and population analysis.

genomics↗